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BIOLOGY OF THE SYNDECANS: A Family of Transmembrane Heparan Sulfate Proteoglycans Merton Bernfield, Robert Kokenyesi, Masato Kato, Michael T. Hinkes, Jiirg Spring, Richard L. Gallo, and Edward J. Lose Joint Program in Neonatology, Harvard Medical School, Boston, Massachusetts 02115 KEY WORDS:
epithelial-mesenchymal interactions, glycosaminoglycans, growth factor re ceptors, matrix receptors, virus receptors
CONTENTS INTRODUCTION . . . .......... .... . .... . .... ................ . .... . . ... . . ...... . . ........... ........ . . .
366
A PRIMER ON HEPARAN SULFATE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .... . . . . . . Structure and Distribution .. ... . . . . . . . . . . . . . . ..... . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . .. . . . . . . . ....
367
.
367 368 370
THE SYNDECAN FAMILY OF CELL SURFACE PROTEOGLYCANS . . . .. . . . . . ....
. .
371
FUNCTIONS OF THE SYNDECANS . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . ... . . . . . . . Matrix Receptor . . .. .. . . . . ..
. .
376 376 377 378 379 380
Protein-binding Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Heparan Sulfate Proteoglyeans at the Cell Surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . .
. . . . .
. . . .
.
.
. . . . . . . . . . . . . . . . . . . .
Organization of Epithelia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Non-thrombogenic Vascular Endothelial Cell Surfaces . . . . .... . . . . .. . . . . . . . . . . . . . . . . . . . . . . Partner in bFGF Action .. .... . . . . . . .. . . ... . . ..... .... . .... . .... . . ... . . .... . . . . . . . . . . . . . . . .... . . Syndecans as Co-receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ALTERATIONS IN SYNDECAN STRUCTURE AND EXPRESSION . . . . . . .. . . . . . . . . . . .
.
.
382 382 384 385
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
386
.
������;:�}rj :;:���::��:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::
i Regulation of Syndecan Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONCLUSIONS
365
0743-4634/92/1115-0365$02.00
366
BERNFIELD ET AL
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INTRODUCTION The cellular microenvironment contains a panoply of insoluble structural as well as diffusible effector molecules that interact with cell surface receptors that signal cells to change behavior . Although replete with highly specific protein receptors, cell surfaces also contain much complex carbohydrate. Indeed, all adherent cells contain glycosaminoglycans (GAG) at their sur faces . Of these GAGs, heparan sulfate (HS) and chondroitin sulfate exist as proteoglycans (PGs), covalently bound to a variety of core proteins . Heparan sulfate is the most ubiquitous cell surface GAG, and it varies in amount during development and following neoplastic transformation. Heparan sulfate is closely related structurally to the more familiar heparin, a GAG that exists in vivo solely as a PG within the granules of mast cells and basophils . Commercial heparin, a protein-free pharmaceutical degradation product of this PG, is used clinically as an anticoagulant. Since this heparin binds to a large number of proteins, it is also used as a chromatographic adsorbent for protein purification. When added to cells in vitro, heparin inhibits a variety of processes, including epithelial glandular morphogenesis (Bemfield 1984), retroviral replication (Baba et al 1988), and smooth muscle cell growth (Guyton et al 1 980). Because there is no qualitative difference between heparin and HS in structure and binding properties (Kjellen & Lindahl 1 991), the protein binding and biological effects of heparin i mplicate HS in diverse cellular processes . Although the ubiquitous presence of heparan sulfate proteoglycans (HSPG) at the cell surface has long been apparent, recent interest in these molecules stems from increasing awareness of the functional implications of their in teractions and the structural characterization of their core proteins . The initial cell surface HSPG to be so characterized was termed syndecan, from the Greek, syndein, to bind together (Saunders et al 1 989a). It bound growth factor and matrix molecules extracellularly and associated with the actin containing cytoskeleton intracellularly , and its expression was highly reg ulated (reviewed in Rapraeger et al 1 987; Bemfield & Sanderson 1 990) . Multiple distinct membrane HSPGs were found in fetal lung fibroblasts (Lories et al 1 987, 1 989) . Molecular cloning then identified one HSPG as similar to syndecan in sequence (Marynen et al 1989), and recently cDNAs for two other HSPG core proteins that share structural features with the syndecan core protein have been described (Baciu et al 199 1; Kojima et al 1 992b; Gould et al 1 992; Carey et al 1 992). Where studied, these PGs also show the tissue-specific and developmental regulation that had been documented for syndecan. These molecules constitute a newly defined family of transmembrane HSPGs that appears to regulate cellular responses to the microenvironment and is the focus of this review. For general discussions of
THE SYNDECANS
367
GAGs and PGs, see the excellent recent reviews by Ruoslahti (1989), Gal
lagher ( 1 989) , Kjellen & Lindahl ( 1 99 1 ) , Esko (1991), and Jackson et al ( 1991). A PRIMER ON HEPARAN SULFATE
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Structure and Distribution Heparan sulfate , initially distinguished from heparin by a difference in extent of sulfation (Jorpes & Gardell 1948), contains greater structural variability than any other GAG (see Figure 1 ) . Both HS and chondroitin sulfate are bound to serine residues in proteins , but a universal consensus sequence for GAG attachment is not known . However, two sequences that commonly attach GAG chains are a ser-gly repeat followed within ca. 5 amino acids by acidic amino acid residues, e.g. (SGMxh-sDfE, found initially in the PG sergJycin (Bourdon et al 1985), and a single ser-gly that is preceded and often flanked by acidic residues, e . g. DfExSG(x)nDfE, found initially in syndecan
HEPARAN SULFATE
.6,.,.1,1.06-06-060
--
HEPARIN
.,.bo URONIC ACIDS:
° •
GlcUA IdoUA
Figure 1
1, • , " .
GLUCOSAMINES:
t::" A
oj
GlcNAc GlcNSulfate
.
Mr (x103) 15-75
5-15
.
ESTER SULFATES:
�"""""6
'�/2
GAG Chains
L�
OR 3
LINKAGE:
°V9 Gal
Xyl
CORE PROTEIN:
I.er
Schematized structures of heparan sulfate and heparin. The HS chain contains a
tetrasaccharide that links the core protein to a linear assembly of alternating uronic acid and N-substituted glucosamine residues. The chain is initially synthesized as a copolymer of D glucuronic acid and N-acetyl glucosamine, which is then modified by sequential enzymatic deacetylation, N-sulfation, and C5 epimerization of D-glucuronate to L-iduronate, and 0sulfation at various positions, which yield chains with considerable sequence diversity, primarily the result of variable sulfation (see Lindahl et al 1986 for review). This sequence heterogeneity, in which each chain is likely distinct, presumably arises because the product of each modification reaction is the substrate for the next and the reactions do not go to completion. Commercial heparin, a pharmaceutical degradation product, contains fragments of chains, the same overall structure and heterogeneity as HS, but rich in L-iduronate, N-sulfate, and O-sulfate. Heparan sulfate contains non-sulfated regions interspersed with these heparin-like highly sulfated regions. It is unclear how the size and distribution of these regions are regulated.
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368
BERNFIELD ET AL
(Saunders et al 1 989a). Variations in the serglycin- and syndecan-types of attachment sites exist, and the precise protein structures required for chain initiation (xylosylation of serine residues) and polymer formation (HS or chondroitin sulfate chains) are not known (Bourdon et al 1 987; Mann et al 1 990). Heparan sulfates and heparin contain the most negatively charged struc tures produced by vertebrate cells and are widely distributed throughout the animal phyla . In addition to the chordates, HS can be found in the mollusks, annelids, and arthropods, including arachnids , crustaceans, and insects , and in organisms as ancient as coelenterates (Nader et al 1988). Heparan sulfate is found intracellularly principally within storage vesicles of various secretory cells and possibly in the nucleus , at the cell surface as part of integral membrane PGs, and extracellularly in the pericellular matrix and basement membrane. At these various sites, HS is generally attached to different core proteins and may have distinct chain size and disaccharide composition. Therefore, the heparin PG that accumulates within the granules of mast cells and basophils can be considered to be one of the large class of HSPGs (Kjellen & Lindahl 1 99 1 ) . Protein-binding Properties Heparan sulfates bind diverse proteins found in the cellular microenviron ment, including extracellular matrix components, peptide growth factors, cell adhesion molecules, lipolytic enzymes , protease inhibitors , and circulating lipoproteins (Table 1 ) . Some of these interactions are required for the function of the protein. Other proteins bound include viral coat proteins, nucleases , DNA and RNA polymerases, and transcription factors. While the HS polyan ion could mimic the interactions of nucleic acids with proteins, the presence in the cellular microenvironment of so many proteins that bind heparinlHS is not likely fortuitous. Heparan sulfate binds proteins at relatively high affinity, with Kd as low as 1-5 nM . The binding is predominantly via electrostatic interactions between the highly anionic sulfate groups on the GAG and clusters of basic amino acids arranged in a three-dimensional array on the protein. Hydrogen bonding involving carboxylate groups may also be involved, and there is a high degree of cooperativity among the anionic sites. This affinity for proteins is in large part due to the L-iduronate residues, which are thought to impart con formational flexibility to the chain (Sanderson et al 1 987) . Three iduronate conformations are in equilibrium within the chain, potentially allowing the iduronate-containing regions to alter their shape and the spatial orientation of the sulfate residues (Casu et aI 1 988). While the local conformation of the HS chain may be altered by interaction with proteins, proteins can change their
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Table 1
Binding interactions of heparinlheparan sulfate (incomplete listing)
Matrix components
Growth factors
Fibronectin wnt-la Interstitial collagen types I, III, V Laminin Pleiotropinb Tenascinc Thrombospondin Vitronectin
FGF family N-CAM aFGF, bFGF, KGF hstIK-fgf, int-2 FGF-5, FGF-6 HB-EGFd HGF/Spe Interferon yf PDGF SDGP VEGFIVPFh
Cell adhesion Degradative enzymes molecules Acetylcholinesterase j Extracellular superoxide dismutase Thrombin Tissue plasminogen activator'
Abbreviations and citations. Except where noted, summarized from Farooqui
Lipolytic enzymes
Protease inhibitors Antithrombin III Heparin cofactor II Leuserpin Plasminogen activator inhibitor-I' Protease nexin I
& Horrockss
1984, Jackson et
Viral coat proteins
Nuclear Lipoproteins proteins
apoB Cholesterol esterase apoE Hepatic & pancreatic triglyceride lipase Lipoprotein lipase
gC and gB of c-fos HSV types I c-jun and II RNA & DNA polymerases gC-II of CMVm gp 120 of HIV Steroid receptors
al 1991, and Kjellen & Lindahl 1991. Bradley & Brown 1990; b also known •
as heparin-binding neurotrophic factor, Merenmies & Rauvala 1990; 'Salmivirta e t al 1991; 'heparin-binding epidermal growth factor, Higashiyama et al 1991; 'hepatocyte growth factor/scatter factor, Tashiro et al 1990; fLortat-Jacob et al 1990; 'Schwannoma-derived growth factor, also known as amphiregulin, Kimura et al 1990; hvascular endothelia growth factorfvascular permeability factor, Ferrara
& Henzel 1989; i Berman
et al 1987; iBrandan et al 1985; 'Andrade-Gordon
& Strickland
1990; I Ehrlich et al 1991;
m
Kari
& Gehrz
1992.
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370
BERNFIELD ET AL
conformation upon binding heparin/HS (for review , see Jackson et al 1991). For example, fibronectin, antithrombin III, and bFGF each undergo a con formational change when they bind heparin. Proteoglycans containing several HS chains may bind to a protein, while the individual HS chains may not, presumably because multiple HS chains interact (Koda & Bemfield 1984). With a single exception, the HS sequences involved in protein binding are not known to be unique, possibly because the needed sequence information is not yet readily available. The exception is the high affinity binding of antithrombin III to a specific pentasaccharide sequence in HS (and in hepa rin), which indicates that binding can be sequence-specific (Marcum et al 1986). Commercial heparin contains distinct sub-populations that bind with high affinity to antithrombin III and to fibronectin (Lee & Lander 1991). Yet, in some interactions of heparin with proteins (e . g . aFGF and bFGF) , all heparin molecules bind with nearly equal affinity, which suggests that a specific sequence is not likely required (Lee & Lander 1991). The nature of the protein-binding site does not necessarily reflect the binding affinity. For example, the HS binding sites on antithrombin III, fibronectin, and bFGF differ substantially in sequence and potentially in shape, but only slightly in binding affinity, all approximating 1 nM (Jackson et al 199 1 ) . HS chains can also self-associate into multimers. The most extensive aggregation occurs between HS chains derived from the same tissue (Fransson 1983). Heparan Sulfate Proteoglycans at the Cell Surface
Heparan sulfate has been known to exist at the cell surface since Kraemer (1971) showed that HS is on several types of adherent cultured cells. Sub sequently, it became clear that some HSPGs could be removed from cells with high salt concentrations , while some were lipophilic , and that HSPGs are the most common PGs in the plasma membrane (Hook et al 1984). Peripheral membrane HSPGs interact via their core proteins with inositol phosphate (Gallagher 1989), or putative receptors (Clement et al 1989), or, less com monly, via their HS chains (Kjellen et al 1 980). Integral HSPGs are bound to the cell surface either via a linkage with membrane phospholipid, as in the HSPG glypican (David et al 1990) and possibly other PGs released by phosphatidyl inositol-specific phospholipase C (Carey & Stahl 1990), or via a hydrophobic transmembrane domain (Kjellen et al 1981). Recognition of these cell surface components led to the notion that they could immobilize proteins at the cell surface (Hook et al 1984) and that they were metabolized actively (Yanagishita & Hascall 1984). These PGs are placed on the cell surface via fusion of vesicles with the plasma membrane and lost from the cell surface both by release into the pericellular environment and by internaliza tion via endocytosis . While all integral membrane PGs are mobile within the
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THE SYNDECANS
371
plane of the membrane , only the transmembrane PGs likely interact with the intracellular cytoskeleton. The transmembrane HSPGs include the syndecan family, betaglycan (or TGF- f3 type III receptor) (Wang et al 1991; Lopez-Casillas et al 1991), and C D-44 (or Hermes antigen, PgP- l or ECMR III) (Brown et al 199 1 ) . These differ in core protein amino acid sequence, but have a similar overall struc ture . Of these, only betaglycan and CD44 exist as variants containing no GAG chains . The mature core proteins contain an extracellular domain of variable length with attachment sites for GAGs and N-linked sugars and, in the syndecans and betaglycan, a putative proteolytic cleavage site near the hydrophobic transmembrane domain, as well as a relatively short (less than 75 residues) cytoplasmic domain . THE SYNDECAN FAMILY OF CELL SURFACE PROTEOGLYCANS Since the molecular cloning of the syndecan core protein from mouse mam mary epithelia (Saunders et al 1 989a) , cDNA-derived amino acid sequences have become available for other PG core proteins that are sufficiently similar to indicate common ancestry . These proteins , plus that of glypican, account for all the membrane HSPG core proteins found in fetal lung fibroblasts (David et aI 1990). These proteins constitute the syndecan family and are here distinguished by number based on the first member to be identified (Table 2). The proteins have a similar domain structure , highly conserved sequences , and a conserved exon organization in the genes studied to date . The syndecan1 gene maps to human chromosome 2p23 (Ala-Kapee et al 1990) and to the syntenic region in the mouse on chromosome 12 (Oettinger et al 1991), while the syndecan-2 gene maps to human chromosome 8q23 (Marynen et al 1989) . Where studied, the core proteins of the syndecan family have similar chemical properties. Each is a HSPG, while syndecan- l also contains chon droitin sulfate (Rapraeger et al 1985; David & Van den Berghe 1985). Chick syndecan-3 has also been proposed to contain chondroitin sulfate (Gould et al 1992). The proteins of both syndecan- l and -2 can form dimers and possibly larger muItimers that resist treatment with sodium dodecyl sulfate (SDS) (Pierce et al 1992; Sanderson et al 1992a; G. David, personal communica tion). Formation of multimers is not the result of interactions between GAG chains or the hydrophobic regions , or disulfide bond formation because apparent multimers can be seen with the GAG-free extracellular domain of syndecan-l , which like syndecan-2, does not contain cysteine . The syndecan proteins behave anomalously on SDS-PAGE possibly because of an extended conformation due to their relatively high proline content . The calculated Mr
372
BERNFIELD ET AL
Table 2
Syndecan family of proteoglycan core proteins
Origin of cDNA
Original designation
Reference
Syndecan-l
mouse mammary epithelia
syndecan
Saunders et al 1989a
human mammary epithelia
syndecan
Mali et al 1990
hamster kidney (BHK-21) fibroblasts
FGF-binding HSPG
Kiefer et al 1990
rat microvascular endothelia
syndecan
Kojima et al 1992b
rat aorta
syndecan
Cizmeci-Smith et al 1992
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Syndecan-2 48KS
Marynen et al 1989
fibroglycan
David et al 1990
mouse 14 day embryos
fibroglycan
G. David,
rat liver
major rat liver membrane
Pierce et al 1992
human fetal lung fibroblasts
personal communication HSPG Syndecan-3 chick embryo limb buds
syndecan-3
Gould et al 1992
rat newborn Schwann cells
N-syndecan
Carey et al 1992
syndecan-4
Baciu et al 199 1 ;
Syndecan-4
chick embryo limb buds
P. Goetinck, personal communication rat microvascular endothelia
ryudocan
Kojima et al 1 992b
for the mature core proteins of s yndecan- l , -2, and -4 is 30.6, 20. 2, and 19.5 kd, while t hey migrate at 69, 48 and 30 kd, respectively . This anomalous m igration is not caused by glycosylation because it remains following chemi cal de-glycosylation of syndecan- l (Weitzhandler et al 1 988) in the in vitro translation products of syndecan- l and syndecan-4 cDNAs (Saunders et al 1989a; Kojima et a1 1992b) and in syndecan- l protein produced in E. coli (R. Kokenyesi, unpublished). Evolution of the syndecans from a common ancestor appears to have m aintained the location and nature of the putative GAG attachment sites , the protease susceptible site adjacent to t he plasma membrane, and the transmem brane and cytoplasmic domains (Figures 2, 3). Size, GAG attachment sites , and sequences indicate a closer structural relationship between the proteins of syndecan-l and -3 (30% sequence identity; rat), and syndecan-2 and -4 (38% sequence identity; rat). In the mouse syndecan- l gene and in the rat and chick syndecan-3 genes , the N-terminal GAG attachment region is encoded by a separate small exon (Hinkes et al 199 1; M . Jalkanen, personal com municat ion; Gould et al 1 992; D. Carey, personal communication) . The additional GAG attachment sites in syndecan- l and -3 are near the plasma
THE SYNDECANS
Syndecan-1
"-IZ"-'?J'--
373
---i� >..: � ...,�..:o:>':o.::. �·:."'"·it ·:"" "":/ I
_________ ___
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DRKE
,ESGX(12)E 1�\:0r::';y;,:·:/1 1-
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Syndecan-3
123 Signal Sequence o Extracellular • Thr-Ser-Pro Rich Region
ERKE
� Transmembrane C] Cytoplasmic
Potential sites: GAG Attachment ... Protease Cleavage
Figure 2 Diagrams showing the derived core protein domain structures, location, and sequences of putative GAG attachment and protease cleavage sites of the syndecans. The proteins are aligned at the start of the transmembrane domain. Shown are syndecan- l (mouse, 3 1 1 amino acids; Saunders et aI 1989a), syndecan-3 (chick, >384 amino acids; Gould et aI 1992) syndecan2 (human, 20 1 amino acids; Marynen et aI1989), and syndecan-4 (rat, 202 amino acids; Kojima et aI1992b). Each protein contains a cluster of similar putative GAG attachment sites distal from the plasma membrane near the N-terminus of the mature protein: one site (or two in syndecan-3) is of the syndecan-type sequence and the other two (or three) sites are of the serglycin-type. An SA site is included for syndecan-2, as previously suggested (Marynen et al 1989). Syndecan-l and -3 contain putative GAG attachment sites that are absent in syndecan-2 and -4. The proposed protease susceptible site adjacent to the transmembrane domain is highly conserved. The se quences of the C-terminal region of the rat proteins are shown in Figure 3.
membrane, are both syndecan-type in sequence , and are e ncoded by separate exons . The more N-terminal of these sites is encoded by a relatively large exon in the rat and chick syndecan-3 gene , which in part resembles the T- , So, and P-rich regions in mucin-like proteins . These sites are not uniformly substituted on syndecan-l from mouse mammary epithelial (NMuMG) cells . Most of the molecules contain only chondroitin sulfate chains at these s ites , but a minor but significant proportion contains both chondroitin sulfate and HS chains (R. Kokenyesi , unpublished). The regions C-terminal to the conserved putative protease-susceptible site are most highly conserved (Figure 3). A single exon in the mouse syndecan- l
374
BERNFIELD ET AL
Syndecan-1
L
Y
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Syndecan-3 Syndecan-2
K RfT]E V L A A V I A G G V[DG F L F A[DF LrJL L L V Y
Syndecan-4
E Rl.:JE
v
Syndecan-1
R M KKK
Syndecan-3
R M KKK D E G S Y T
L A A L I�G G
v v
G I L F AV F
L
W
L
L LV Y
F YA
D E G
Syndecan-2 Syndecan-4
Figure 3
R M KKK
DE G S Y D L G -
Derived amino acid sequence of the highly conserved C-terminal regions of the rat
syndecan core proteins, which show the region containing the putative protease susceptible site, the transmembrane domain, and the cytoplasmic domain (Cizmeci-Smith et al 1992; Pierce et al 1992; Carey et al 1992; Kojima et al 1992b). The amino acids conserved in the sequences are
maximal identity, syndecans-I and -3 (70%) and syndecans-2 and -4 (77%) are adjacent to each other. The putative protease sites (bold) eontain two basic residues in syndecans-I, -2 anmd -3, but solely arginine in syndecan-4. The transmembrane domains and the stop transfer sequence are of the same length in each protein, within the box in the diagram; the sequences showing
while the remainder of the cytoplasmic domains ean be aligned only by introducing several
independent deletions. A tyrosine (bold) at the end of the transmembrane domain and three tyrosines (bold) in the cytoplasmic domain are conserved in each protein. The loop between the first and the second tyrosin e of the cytoplasmic domain is the most disparate region of this domain
and varies in sequence and in length. This region and the loop between the second and third tyrosine of syndecan - l and -3 are quite similar, as are these regions in syndecan-2 and -4.
gene and in the rat and chick syndecan-3 genes encodes the identical portion of this region. The tyrosine that completes the transmembrane domain and the three tyrosines in the cytoplasmic domain are invariant. The length and sequence between the transmembrane domain and the first tyrosine are con served and could account, in syndecan-l and -3, for a tyrosine internalization signal (Ktistakis et al 1990). However, the distance between the next tyro sines differs,
possibly providing individual syndecans with
specificity
towards interacting proteins. One of the tyrosines fits a consensus sequence for tyrosine phosphorylation (Gould et al 1992; Pierce et al 1992). Comparisons between species of derived protein sequences for the syn decans show an intriguing evolutionary relationship (Figure 4). Syndecan-l sequences of the mouse (Saunders et al 1989a), human (Mali et al 1990), hamster (Kiefer et al 1990), and rat proteins (Cizmeci-Smith et al 1992;
THE SYNDECANS
100%
Rat
l?dl
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I� I II
88%
III I I 1111111 II II
@
I
1111111 11I1II
82%
III lUI II I
���y
III
F0JX?:?:?/ 100% 100%
yyyy
� Signal Sequence m Transmembrane
I
96% 100%
96% 100%
70%
91%
Human
I I
If'�1!X/Ad ���� I II I I f?jll 111111111111111111111111111111 11 [ 111�/:}\:: '.: 1
95%
Hamster
II
II
Mouse
375
y���
flliJ Cytoplasmic
o
Extracellular
Potential sites:
I
GAG Attachment
1 Protease Cleavage
Figure 4 Diagram showing the core protein domain structure and sequence conservation of syndecan-I derived from the mouse (Saunders et al 1989a), rat (Cizmeci-Smith et al 1992; Kojima et al 1992b), hamster (Kiefer et al 1990), and human (Mali et al 1990) cDNAs. Percent identity to the mouse protein is shown above each domain and the black lines within a domain I (bar) indicate the position of the amino acids that differ from the mouse protein sequence. Lines above the bars indicate the putative glycosaminoglycan attachment sites. The locations of the invariant tyrosines are also shown.
Kojima et al 1 992b) show that the cytoplasmic domain and , not unexpectedly, the transmembrane domain do not evolve among these species, but the extracellular domain is evolving extremely rapidly, except for highly con
served sequences surrounding the GAG attachment sites and at the putative A nearly identical pattern of conservation is seen in the mouse , human , and rat syndecan-2 proteins. The extracellular domains of human and mouse syndecan-1 show only 70% sequence identity and thus these domains diverge as rapidly as plasma albumin and lysozyme , proteins with very rapidly evolving protein sequences (Doolittle 1992) . Moreover , the available rat and chick syndecan- 3 and rat and chick syndecan-4 sequences are 57 and 52% identical, respectively, which ranks them among the fastest changing vertebrate proteins (Doolittle 1 992) . The sequence comparisons suggest that the functional elements of the extracellular domain are the GAG chains and the protease susceptible sites near the plasma membrane . Moreover, the cytoplasmic domain likely inprotease-susceptible site.
376
B ERNFIELD ET AL
teracts with evolutionarily stable components such as cytoskeletal (e.g. actin, tubulins) and regulatory (e.g. calmodulin) proteins. Distinct evolutionary stability of extracellular and cytoplasmic domains is also seen in the tyrosine kinase and tyrosine phosphatase families of membrane-spanning proteins (Doolittle 1991). These, like syndecan-l , function as receptors at the cell surface.
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FUNCTIONS OF THE SYNDECANS Matrix Receptor All adherent cells detect and respond to the extracellular matrix, an insoluble amalgam of large multidomain proteins. The matrix produced by mesenchy mal cells accumulates in the interstitial spaces, whereas the matrix produced by epithelial cells organizes into a basement membrane (for review, see Bemfield et aI1984). Syndecan-l binds cells via its HS chains to a variety of components of the interstitial matrix, including types I, III, and V collagen, the fibrillar collagens (Koda & Bemfield 1984), fibronectin (Saunders & Bemfield 1988), thrombospondin (Sun et al 1989), and tenascin (Salmivirta et al 1991). This binding is independent of calcium and magnesium ions (Koda et al1985; Saunders & Bemfield1988) and is abolished by trypsin treatment of cells, thus distinguishing it from matrix binding via the integrins, a family of heterodimeric proteins receptors (Albelda & Buck 1990). At physiological pH and ionic strength, similar binding is not seen with the protein-free GAG chains isolated from syndecan-l of NMuMG cells. Under these conditions, the PG binds with relatively high affinity (Kd for type I collagen fibrils is ca. 1 nM) (Koda & Bemfield 1984), but binds to neither laminin nor type IV collagen, both components of the epithelial basement membrane (Koda et al 1985; Elenius et al 1990). Binding to collagens appears to require the fibril; stochiometry is 2-300 type 1 collagen monomers per each syndecan-l mole cule, varying with the fibril preparation, and no binding is seen to denatured collagen or to individual collagen monomers (Koda & Bemfield 1984; Koda et al 1985). Although these binding properties alone are not sufficient to implicate syndecan-l as a physiologically relevant matrix receptor, its expression is also consistent with this role. It polarizes to the basolateral surfaces of simple epithelia, both in culture (Rapraeger et al 1987) and in tissues (Hayashi et al 1987), and localizes in early mouse embryos to the site of initial matrix accumulation (Sutherland et al 1991). Syndecan-l binds cells of the B-cell lineage to type I collagen (Sanderson et al 1992b), and its expression corre lates with the sites and times that B cells associate with matrix (Sanderson et al 1989): It is expressed on precursor B cells in the bone marrow, but not after the cells mature and are released into the circulation as lymphocytes. Syn-
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decan- l reappears when the cells differentiate into plasma cells in lymphoid tissues. Other properties also implicate cell surlace syndecan-l as a matrix recep tor. It is shed within minutes from polarized NMuMG cells when they are suspended and is not replaced on the cell surlace until these cells re-attach to a substratum (Jalkanen et al 1987 ) . While most syndecan-l in non-polarized, mammary epithelia is detergent-extractable, which suggests that it is free in the membrane, cross-linking of cell surlace syndecan- l by antibodies or matrix molecules induces it to associate with the actin-containing cytoskele ton and resist detergent extraction (Rapraeger et aI 1 987) . Indeed, syndecan- l and F-actin filaments co-localize at basal cell surlaces in polarized mammary epithelia (Rapraeger et al 1 987; Kato & Bemfield 1 990), and isolated syn decan- l , but not its extracellular domain, binds directly or indirectly to F-actin filaments in a sedimentation assay (Rapraeger & Bemfield 1 982) . Thus, syndecan-l may link the matrix to the cytoskeleton. These results are most consistent with syndecan- l acting as a matrix receptor, at least for epithelia where it is predominantly found in mature organisms. Because of its high abundance (ca. 1 06 syndecan- l molecules per NMuMG cell surlace; M. Bemfield, unpublished) and its relatively high binding affinity, it is potentially important that syndecan-l is cleaved from the plasma membrane when epithelial cells are induced to change shape. This mechanism enables rapid and possibly controlled release of the cells from the matrix, which accommodates cell movements or changes in cell shape during morphogenesis and tissue repair . Although its extended conformation might enable the HS chains to interact, its binding preference for interstitial matrix rather than basement membrane components is inconsistent with this role for epithelia. Syndecan- l also localizes to cell surlaces where there is no matrix apparent , e.g. at the lateral surlaces of simple epithelia and over the entire surface of stratified epithelia, and is markedly diminished in amount and not concentrated at adhesive sites on mesenchymal cell surlaces (Kato & Bem field 1 989) . These distributions suggest additional functions for syndecan- l . Organization of Epithelia
Syndecan-l expression correlates with normal epithelial cell organization in vitro. Studies with a mouse mammary tumor cell line (S 1 15 cells) that lose an epithelioid morphology and become fusiform in response to steroid hormones (Yates & King 1 98 1 ) i mplicate syndecan- l in organizing epithelial cells into sheets (Leppa et al 1 99 1 ) . When these cells are induced to change shape by testosterone, the levels of cell surlace syndecan- l and mRNA fall. Glucocor ticoids also cause these cells to become fusiform. When these cells are transfected with a syndecan- l cDNA under the control of a glucocorticoid responsive MMTV-LTR, hormone-induced expression of syndecan- l pre vents the change in morphology. While the parental S115 cells, similar to
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other carcinoma cells, grow on a soft agar substratum, the hormone-treated transfectants fail to grow, which suggests that syndecan- l expression restores at least one aspect of the non-transformed phenotype (Leppii et al 1992) . NMuMG cells made syndecan - l-deficient by stable transfection with the syndecan- 1 coding region in an antisense configuration show two distinct morphologies: clones bearing more than 45% normal levels of cell surface syndecan- l grow as islands of closely adherent epithelia, whereas clones showing less than lO% normal levels grow as individual fusiform cells that overlap each other (Saunders et al 1 989b; Kato & Bemfield 1 990) . Whereas vector-only controls grow as sheets on top of collagen gels and as duct-like structures within it, the fusiform cells invade the gels and migrate within them, mimicking the behavior of authentic mesenchymal cells. The fusiform cells retain the milk fat globule antigen and cytokeratins, and thus have mammary epithelial characteristics, but lose an epithelia-like organized actin cytoskeleton, redistribute 13 1 integrins , and markedly decrease E-cadherin expression. The cytoskeletal, 13 1 integrin , and E-cadherin changes in syndecan- 1 deficient cells may be a consequence of the change i n cell shape resulting from loss of a matrix receptor. However , syndecan- l co-localizes with F-actin bundles at the lateral surfaces of NMuMG cells (Rapraeger et al 1987). The se bundles are apparently associated with the zonula adherens, the cellular organelle responsible for epithelial organization. Certain 13 1 integrins and E-cadherin, the major epithelial cell adhesion molecule (Takeichi 1 99 1 ) , are also found at this site . Thus, syndecan- l may participate in a membrane cytoskeletal complex of adhesion proteins that maintains the organization of epithelial sheets . Such a complex may exist at various sites within epithelial cells, which enables syndecan- 1 to be a member of a matrix receptor complex at the basal cell surface and an adhesion complex at adherens junctions. A role for syndecan- 1 in maintaining epithelial organization is also sug gested by its expression during epithelial-mesenchymal transformations in embryos . During development of the secondary palate in rodents, the medial edge epithelia of the nasal and maxillary processes transform into mesen chymal cells (Fitchett et al 1 990). Immediately prior to this transformation and precisely at the sites where the epithelia will become fusiform cells, syndecan- 1 expression is markedly reduced , and a decrease in E-cadherin expression follows (Fitchett et al 1 990, K . McAlmon, unpublished) . The intermediate filaments then switch from cytokeratins to vimentin and these now fusiform cells migrate into the interstitium. Non-thrombogenic Vascular Endothelial Cell Surfaces
Heparin has long been used clinically as an anticoagulant . The heparin-like GAGs promote anticoagulation by interacting with the proteases of the in trinsic coagulation cascade and thrombin, as well as a variety of inhibitors,
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including protein C and the serpins, a family of serine protease inhibitors that includes antithrombin Ill . A specific pentasaccharide sequence found in heparin and HS binds to antithrombin III, thereby enabling it to form an enzymatically inactive complex with several coagulation enzymes (Daniels son et al 1986) . A similar interaction with protease nexins promotes the inactivation of pericellular proteases (Farrell & Cunningham 1986) . Anticoagulantly active HS on vascular endothelial cell surfaces could endow them with non-thrombogenic properties (Colburn & Buonassisi 1 982). Rat fat pad microvascular endothelial cells produce such HS chains, which accounts for less than 5% of their total HSPG (Kojima et al 1992a). While a low proportion , this is five- to tenfold more anticoagulant HS than produced by bovine aortic endothelial cells (Marcum et aI 1 986) . Importantly, these HS chains are not on a single type of core protein , but are on syndecan- l and -4 and possibly other cell surface PGs (Kojima et al 1992a). On average, each anticoagulantly active HS chain contains a single interacting site. Therefore, at least in these cells , the primary sequences of the different syndecan core proteins do not dictate enrichment of their HS chains for the distinct anti thrombin III binding structure. These microvascular endothelial cells contain more syndecan- l than syn decan-4 mRNA and protein , while rat aortic smooth muscle cells and rat fat pad adipocytes (Kojima et al 1992b) contain more syndecan-4 mRNA . Syn decan- l in bovine pulmonary artery endothelial cells is primarily an in tracellular molecule, as in other mesenchymal cells . The small proportion on the cell surface localizes predominantly at the basal surface , the putative abluminal surface (Lose & Bernfield 199 1 ) . Thus, other HSPGs may also be involved in producing a non-thrombogenic luminal surface. Partner in bFGF Action
Fibroblast growth factors (FGF) are a family of heparin-binding growth factors that promote the proliferation and migration of mesenchymal cells and the survival and differentiation of neural cells . Currently, there are seven members of this family, and these have a variety of sources and biological roles , including angiogenesis , embryonic inductions , repair of injury, and neoplastic transformation (see Burgess & Maciag 1989 for review) . Cell surface HSPGs are apparently required for the bFGF action and may be involved in the action of other members of the FGF family (Klagsbrun & Baird 199 1 ) . The binding of bFGF to heparin and to HSPG in the basement membrane and at the cell surface is thought to be involved in bFGF activity because mutation of the heparin/HS binding site on bFGF reduces its biologi cal activity (Seno et al 1 990) . The activity of acidic FGF is also stabilized by heparin and, at least in a parathyroid cell line, requires a cell surface HSPG (Sakaguchi et al 199 1 ) . Cell surface H S appears to be required for the binding of bFGF to its high
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affinity receptor . CHO cells defective in HS synthesis do not bind bFGF even after inducing the expression of FGFR- l , a signal-transducing high affinity bFGF receptor . They will bind, however , when exogenous heparin is avail able, which suggests that binding to cell surface HS is a prerequisite for high affinity binding (Yayon et al 1 99 1 ) . This work was extended by findings that cells made deficient in cell surface HS , by heparitinase pretreatment , or by chlorate treatment to prevent sulfation, have a markedly decreased ability to respond to bFGF as assessed by proliferation (Swiss 3T3 cells), or by repression of terminal differentiation (MM14 myoblasts) (Rapraeger et al 1 99 1 ) . These studies do not identify a specific cell surface HSPG as the low affinity receptor for bFGF but syndecans are prime candidates. Syndecan- l from mammary epithelial cells binds bFGF via its HS chains (Bem field & Hooper 1 991), and cell surface expression of hamster syndecan-l in l ympho blastoid cells endows these cells with low-affinity binding sites for bFGF (Kiefer et al 1 990) . Moreover , expression of syndecan- l correlates with the potential activities of bFGF or its family members during development (see section on developmental regulation). Syndecan- l expression during early mouse development corresponds with the sites of bFGF action in inducing axial mesoderm formation in Xenopus laevis (Amaya et al 1 99 l ) . Syndecan- l is also expressed transiently on a variety of condensing mesenchymal cell popUlations (e. g . tooth , kidney, limb) when they are rapidly proliferating and is lost when these cells differentiate, consistent with the known effects of bFGF on mesenchymal cells . Syndecans as Co-receptors
Several functions proposed for the syndecans involve binding components of the cellular microenvironment that themselves interact with a cell surface receptor (Figure 5). For example, fibronectin and interstitial collagens bind syndecan- l , but also bind various integrin receptors, and bFGF binds syn decan- l, but also associates with its high affinity protein receptor. Many additional interactions are possible because heparin and HS bind a broad range of matrix components , growth factors , cell adhesion molecules, degra dative enzymes, and protease inhibitors (cf Table 1 ) . For instance , heparin binding EGF also binds the EGF receptor (Higashiyama et al 1 99 1 ), N-CAM also has homophilic interactions (Reyes et al 1 990), and thrombin also interacts with a specific receptor (Pouyssegur & Seuwen 1 992) . The signifi cance of these dual interactions is unclear, but binding to cell surface HS is often not suspected because it is ubiquitous. Thus, cell surface HSPGs may be co-receptors, integral membrane components that are structurally distinct from high affinity receptors , which bind biological ligands and participate in formation of a receptor complex that is required to generate the ligand's physiological action.
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Syndecans
+
F.
"
ANNUAL REVIEWS
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rights reserved
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BIOLOGY OF THE SYNDECANS: A Family of Transmembrane Heparan Sulfate Proteoglycans Merton Bernfield, Robert Kokenyesi, Masato Kato, Michael T. Hinkes, Jiirg Spring, Richard L. Gallo, and Edward J. Lose Joint Program in Neonatology, Harvard Medical School, Boston, Massachusetts 02115 KEY WORDS:
epithelial-mesenchymal interactions, glycosaminoglycans, growth factor re ceptors, matrix receptors, virus receptors
CONTENTS INTRODUCTION . . . .......... .... . .... . .... ................ . .... . . ... . . ...... . . ........... ........ . . .
366
A PRIMER ON HEPARAN SULFATE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .... . . . . . . Structure and Distribution .. ... . . . . . . . . . . . . . . ..... . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . .. . . . . . . . ....
367
.
367 368 370
THE SYNDECAN FAMILY OF CELL SURFACE PROTEOGLYCANS . . . .. . . . . . ....
. .
371
FUNCTIONS OF THE SYNDECANS . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . ... . . . . . . . Matrix Receptor . . .. .. . . . . ..
. .
376 376 377 378 379 380
Protein-binding Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Heparan Sulfate Proteoglyeans at the Cell Surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . .
. . . . .
. . . .
.
.
. . . . . . . . . . . . . . . . . . . .
Organization of Epithelia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Non-thrombogenic Vascular Endothelial Cell Surfaces . . . . .... . . . . .. . . . . . . . . . . . . . . . . . . . . . . Partner in bFGF Action .. .... . . . . . . .. . . ... . . ..... .... . .... . .... . . ... . . .... . . . . . . . . . . . . . . . .... . . Syndecans as Co-receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ALTERATIONS IN SYNDECAN STRUCTURE AND EXPRESSION . . . . . . .. . . . . . . . . . . .
.
.
382 382 384 385
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
386
.
������;:�}rj :;:���::��:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::
i Regulation of Syndecan Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONCLUSIONS
365
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INTRODUCTION The cellular microenvironment contains a panoply of insoluble structural as well as diffusible effector molecules that interact with cell surface receptors that signal cells to change behavior . Although replete with highly specific protein receptors, cell surfaces also contain much complex carbohydrate. Indeed, all adherent cells contain glycosaminoglycans (GAG) at their sur faces . Of these GAGs, heparan sulfate (HS) and chondroitin sulfate exist as proteoglycans (PGs), covalently bound to a variety of core proteins . Heparan sulfate is the most ubiquitous cell surface GAG, and it varies in amount during development and following neoplastic transformation. Heparan sulfate is closely related structurally to the more familiar heparin, a GAG that exists in vivo solely as a PG within the granules of mast cells and basophils . Commercial heparin, a protein-free pharmaceutical degradation product of this PG, is used clinically as an anticoagulant. Since this heparin binds to a large number of proteins, it is also used as a chromatographic adsorbent for protein purification. When added to cells in vitro, heparin inhibits a variety of processes, including epithelial glandular morphogenesis (Bemfield 1984), retroviral replication (Baba et al 1988), and smooth muscle cell growth (Guyton et al 1 980). Because there is no qualitative difference between heparin and HS in structure and binding properties (Kjellen & Lindahl 1 991), the protein binding and biological effects of heparin i mplicate HS in diverse cellular processes . Although the ubiquitous presence of heparan sulfate proteoglycans (HSPG) at the cell surface has long been apparent, recent interest in these molecules stems from increasing awareness of the functional implications of their in teractions and the structural characterization of their core proteins . The initial cell surface HSPG to be so characterized was termed syndecan, from the Greek, syndein, to bind together (Saunders et al 1 989a). It bound growth factor and matrix molecules extracellularly and associated with the actin containing cytoskeleton intracellularly , and its expression was highly reg ulated (reviewed in Rapraeger et al 1 987; Bemfield & Sanderson 1 990) . Multiple distinct membrane HSPGs were found in fetal lung fibroblasts (Lories et al 1 987, 1 989) . Molecular cloning then identified one HSPG as similar to syndecan in sequence (Marynen et al 1989), and recently cDNAs for two other HSPG core proteins that share structural features with the syndecan core protein have been described (Baciu et al 199 1; Kojima et al 1 992b; Gould et al 1 992; Carey et al 1 992). Where studied, these PGs also show the tissue-specific and developmental regulation that had been documented for syndecan. These molecules constitute a newly defined family of transmembrane HSPGs that appears to regulate cellular responses to the microenvironment and is the focus of this review. For general discussions of
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GAGs and PGs, see the excellent recent reviews by Ruoslahti (1989), Gal
lagher ( 1 989) , Kjellen & Lindahl ( 1 99 1 ) , Esko (1991), and Jackson et al ( 1991). A PRIMER ON HEPARAN SULFATE
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Structure and Distribution Heparan sulfate , initially distinguished from heparin by a difference in extent of sulfation (Jorpes & Gardell 1948), contains greater structural variability than any other GAG (see Figure 1 ) . Both HS and chondroitin sulfate are bound to serine residues in proteins , but a universal consensus sequence for GAG attachment is not known . However, two sequences that commonly attach GAG chains are a ser-gly repeat followed within ca. 5 amino acids by acidic amino acid residues, e.g. (SGMxh-sDfE, found initially in the PG sergJycin (Bourdon et al 1985), and a single ser-gly that is preceded and often flanked by acidic residues, e . g. DfExSG(x)nDfE, found initially in syndecan
HEPARAN SULFATE
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GLUCOSAMINES:
t::" A
oj
GlcNAc GlcNSulfate
.
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5-15
.
ESTER SULFATES:
�"""""6
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GAG Chains
L�
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LINKAGE:
°V9 Gal
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CORE PROTEIN:
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Schematized structures of heparan sulfate and heparin. The HS chain contains a
tetrasaccharide that links the core protein to a linear assembly of alternating uronic acid and N-substituted glucosamine residues. The chain is initially synthesized as a copolymer of D glucuronic acid and N-acetyl glucosamine, which is then modified by sequential enzymatic deacetylation, N-sulfation, and C5 epimerization of D-glucuronate to L-iduronate, and 0sulfation at various positions, which yield chains with considerable sequence diversity, primarily the result of variable sulfation (see Lindahl et al 1986 for review). This sequence heterogeneity, in which each chain is likely distinct, presumably arises because the product of each modification reaction is the substrate for the next and the reactions do not go to completion. Commercial heparin, a pharmaceutical degradation product, contains fragments of chains, the same overall structure and heterogeneity as HS, but rich in L-iduronate, N-sulfate, and O-sulfate. Heparan sulfate contains non-sulfated regions interspersed with these heparin-like highly sulfated regions. It is unclear how the size and distribution of these regions are regulated.
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(Saunders et al 1 989a). Variations in the serglycin- and syndecan-types of attachment sites exist, and the precise protein structures required for chain initiation (xylosylation of serine residues) and polymer formation (HS or chondroitin sulfate chains) are not known (Bourdon et al 1 987; Mann et al 1 990). Heparan sulfates and heparin contain the most negatively charged struc tures produced by vertebrate cells and are widely distributed throughout the animal phyla . In addition to the chordates, HS can be found in the mollusks, annelids, and arthropods, including arachnids , crustaceans, and insects , and in organisms as ancient as coelenterates (Nader et al 1988). Heparan sulfate is found intracellularly principally within storage vesicles of various secretory cells and possibly in the nucleus , at the cell surface as part of integral membrane PGs, and extracellularly in the pericellular matrix and basement membrane. At these various sites, HS is generally attached to different core proteins and may have distinct chain size and disaccharide composition. Therefore, the heparin PG that accumulates within the granules of mast cells and basophils can be considered to be one of the large class of HSPGs (Kjellen & Lindahl 1 99 1 ) . Protein-binding Properties Heparan sulfates bind diverse proteins found in the cellular microenviron ment, including extracellular matrix components, peptide growth factors, cell adhesion molecules, lipolytic enzymes , protease inhibitors , and circulating lipoproteins (Table 1 ) . Some of these interactions are required for the function of the protein. Other proteins bound include viral coat proteins, nucleases , DNA and RNA polymerases, and transcription factors. While the HS polyan ion could mimic the interactions of nucleic acids with proteins, the presence in the cellular microenvironment of so many proteins that bind heparinlHS is not likely fortuitous. Heparan sulfate binds proteins at relatively high affinity, with Kd as low as 1-5 nM . The binding is predominantly via electrostatic interactions between the highly anionic sulfate groups on the GAG and clusters of basic amino acids arranged in a three-dimensional array on the protein. Hydrogen bonding involving carboxylate groups may also be involved, and there is a high degree of cooperativity among the anionic sites. This affinity for proteins is in large part due to the L-iduronate residues, which are thought to impart con formational flexibility to the chain (Sanderson et al 1 987) . Three iduronate conformations are in equilibrium within the chain, potentially allowing the iduronate-containing regions to alter their shape and the spatial orientation of the sulfate residues (Casu et aI 1 988). While the local conformation of the HS chain may be altered by interaction with proteins, proteins can change their
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Table 1
Binding interactions of heparinlheparan sulfate (incomplete listing)
Matrix components
Growth factors
Fibronectin wnt-la Interstitial collagen types I, III, V Laminin Pleiotropinb Tenascinc Thrombospondin Vitronectin
FGF family N-CAM aFGF, bFGF, KGF hstIK-fgf, int-2 FGF-5, FGF-6 HB-EGFd HGF/Spe Interferon yf PDGF SDGP VEGFIVPFh
Cell adhesion Degradative enzymes molecules Acetylcholinesterase j Extracellular superoxide dismutase Thrombin Tissue plasminogen activator'
Abbreviations and citations. Except where noted, summarized from Farooqui
Lipolytic enzymes
Protease inhibitors Antithrombin III Heparin cofactor II Leuserpin Plasminogen activator inhibitor-I' Protease nexin I
& Horrockss
1984, Jackson et
Viral coat proteins
Nuclear Lipoproteins proteins
apoB Cholesterol esterase apoE Hepatic & pancreatic triglyceride lipase Lipoprotein lipase
gC and gB of c-fos HSV types I c-jun and II RNA & DNA polymerases gC-II of CMVm gp 120 of HIV Steroid receptors
al 1991, and Kjellen & Lindahl 1991. Bradley & Brown 1990; b also known •
as heparin-binding neurotrophic factor, Merenmies & Rauvala 1990; 'Salmivirta e t al 1991; 'heparin-binding epidermal growth factor, Higashiyama et al 1991; 'hepatocyte growth factor/scatter factor, Tashiro et al 1990; fLortat-Jacob et al 1990; 'Schwannoma-derived growth factor, also known as amphiregulin, Kimura et al 1990; hvascular endothelia growth factorfvascular permeability factor, Ferrara
& Henzel 1989; i Berman
et al 1987; iBrandan et al 1985; 'Andrade-Gordon
& Strickland
1990; I Ehrlich et al 1991;
m
Kari
& Gehrz
1992.
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conformation upon binding heparin/HS (for review , see Jackson et al 1991). For example, fibronectin, antithrombin III, and bFGF each undergo a con formational change when they bind heparin. Proteoglycans containing several HS chains may bind to a protein, while the individual HS chains may not, presumably because multiple HS chains interact (Koda & Bemfield 1984). With a single exception, the HS sequences involved in protein binding are not known to be unique, possibly because the needed sequence information is not yet readily available. The exception is the high affinity binding of antithrombin III to a specific pentasaccharide sequence in HS (and in hepa rin), which indicates that binding can be sequence-specific (Marcum et al 1986). Commercial heparin contains distinct sub-populations that bind with high affinity to antithrombin III and to fibronectin (Lee & Lander 1991). Yet, in some interactions of heparin with proteins (e . g . aFGF and bFGF) , all heparin molecules bind with nearly equal affinity, which suggests that a specific sequence is not likely required (Lee & Lander 1991). The nature of the protein-binding site does not necessarily reflect the binding affinity. For example, the HS binding sites on antithrombin III, fibronectin, and bFGF differ substantially in sequence and potentially in shape, but only slightly in binding affinity, all approximating 1 nM (Jackson et al 199 1 ) . HS chains can also self-associate into multimers. The most extensive aggregation occurs between HS chains derived from the same tissue (Fransson 1983). Heparan Sulfate Proteoglycans at the Cell Surface
Heparan sulfate has been known to exist at the cell surface since Kraemer (1971) showed that HS is on several types of adherent cultured cells. Sub sequently, it became clear that some HSPGs could be removed from cells with high salt concentrations , while some were lipophilic , and that HSPGs are the most common PGs in the plasma membrane (Hook et al 1984). Peripheral membrane HSPGs interact via their core proteins with inositol phosphate (Gallagher 1989), or putative receptors (Clement et al 1989), or, less com monly, via their HS chains (Kjellen et al 1 980). Integral HSPGs are bound to the cell surface either via a linkage with membrane phospholipid, as in the HSPG glypican (David et al 1990) and possibly other PGs released by phosphatidyl inositol-specific phospholipase C (Carey & Stahl 1990), or via a hydrophobic transmembrane domain (Kjellen et al 1981). Recognition of these cell surface components led to the notion that they could immobilize proteins at the cell surface (Hook et al 1984) and that they were metabolized actively (Yanagishita & Hascall 1984). These PGs are placed on the cell surface via fusion of vesicles with the plasma membrane and lost from the cell surface both by release into the pericellular environment and by internaliza tion via endocytosis . While all integral membrane PGs are mobile within the
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THE SYNDECANS
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plane of the membrane , only the transmembrane PGs likely interact with the intracellular cytoskeleton. The transmembrane HSPGs include the syndecan family, betaglycan (or TGF- f3 type III receptor) (Wang et al 1991; Lopez-Casillas et al 1991), and C D-44 (or Hermes antigen, PgP- l or ECMR III) (Brown et al 199 1 ) . These differ in core protein amino acid sequence, but have a similar overall struc ture . Of these, only betaglycan and CD44 exist as variants containing no GAG chains . The mature core proteins contain an extracellular domain of variable length with attachment sites for GAGs and N-linked sugars and, in the syndecans and betaglycan, a putative proteolytic cleavage site near the hydrophobic transmembrane domain, as well as a relatively short (less than 75 residues) cytoplasmic domain . THE SYNDECAN FAMILY OF CELL SURFACE PROTEOGLYCANS Since the molecular cloning of the syndecan core protein from mouse mam mary epithelia (Saunders et al 1 989a) , cDNA-derived amino acid sequences have become available for other PG core proteins that are sufficiently similar to indicate common ancestry . These proteins , plus that of glypican, account for all the membrane HSPG core proteins found in fetal lung fibroblasts (David et aI 1990). These proteins constitute the syndecan family and are here distinguished by number based on the first member to be identified (Table 2). The proteins have a similar domain structure , highly conserved sequences , and a conserved exon organization in the genes studied to date . The syndecan1 gene maps to human chromosome 2p23 (Ala-Kapee et al 1990) and to the syntenic region in the mouse on chromosome 12 (Oettinger et al 1991), while the syndecan-2 gene maps to human chromosome 8q23 (Marynen et al 1989) . Where studied, the core proteins of the syndecan family have similar chemical properties. Each is a HSPG, while syndecan- l also contains chon droitin sulfate (Rapraeger et al 1985; David & Van den Berghe 1985). Chick syndecan-3 has also been proposed to contain chondroitin sulfate (Gould et al 1992). The proteins of both syndecan- l and -2 can form dimers and possibly larger muItimers that resist treatment with sodium dodecyl sulfate (SDS) (Pierce et al 1992; Sanderson et al 1992a; G. David, personal communica tion). Formation of multimers is not the result of interactions between GAG chains or the hydrophobic regions , or disulfide bond formation because apparent multimers can be seen with the GAG-free extracellular domain of syndecan-l , which like syndecan-2, does not contain cysteine . The syndecan proteins behave anomalously on SDS-PAGE possibly because of an extended conformation due to their relatively high proline content . The calculated Mr
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BERNFIELD ET AL
Table 2
Syndecan family of proteoglycan core proteins
Origin of cDNA
Original designation
Reference
Syndecan-l
mouse mammary epithelia
syndecan
Saunders et al 1989a
human mammary epithelia
syndecan
Mali et al 1990
hamster kidney (BHK-21) fibroblasts
FGF-binding HSPG
Kiefer et al 1990
rat microvascular endothelia
syndecan
Kojima et al 1992b
rat aorta
syndecan
Cizmeci-Smith et al 1992
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Syndecan-2 48KS
Marynen et al 1989
fibroglycan
David et al 1990
mouse 14 day embryos
fibroglycan
G. David,
rat liver
major rat liver membrane
Pierce et al 1992
human fetal lung fibroblasts
personal communication HSPG Syndecan-3 chick embryo limb buds
syndecan-3
Gould et al 1992
rat newborn Schwann cells
N-syndecan
Carey et al 1992
syndecan-4
Baciu et al 199 1 ;
Syndecan-4
chick embryo limb buds
P. Goetinck, personal communication rat microvascular endothelia
ryudocan
Kojima et al 1 992b
for the mature core proteins of s yndecan- l , -2, and -4 is 30.6, 20. 2, and 19.5 kd, while t hey migrate at 69, 48 and 30 kd, respectively . This anomalous m igration is not caused by glycosylation because it remains following chemi cal de-glycosylation of syndecan- l (Weitzhandler et al 1 988) in the in vitro translation products of syndecan- l and syndecan-4 cDNAs (Saunders et al 1989a; Kojima et a1 1992b) and in syndecan- l protein produced in E. coli (R. Kokenyesi, unpublished). Evolution of the syndecans from a common ancestor appears to have m aintained the location and nature of the putative GAG attachment sites , the protease susceptible site adjacent to t he plasma membrane, and the transmem brane and cytoplasmic domains (Figures 2, 3). Size, GAG attachment sites , and sequences indicate a closer structural relationship between the proteins of syndecan-l and -3 (30% sequence identity; rat), and syndecan-2 and -4 (38% sequence identity; rat). In the mouse syndecan- l gene and in the rat and chick syndecan-3 genes , the N-terminal GAG attachment region is encoded by a separate small exon (Hinkes et al 199 1; M . Jalkanen, personal com municat ion; Gould et al 1 992; D. Carey, personal communication) . The additional GAG attachment sites in syndecan- l and -3 are near the plasma
THE SYNDECANS
Syndecan-1
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373
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Syndecan-3
123 Signal Sequence o Extracellular • Thr-Ser-Pro Rich Region
ERKE
� Transmembrane C] Cytoplasmic
Potential sites: GAG Attachment ... Protease Cleavage
Figure 2 Diagrams showing the derived core protein domain structures, location, and sequences of putative GAG attachment and protease cleavage sites of the syndecans. The proteins are aligned at the start of the transmembrane domain. Shown are syndecan- l (mouse, 3 1 1 amino acids; Saunders et aI 1989a), syndecan-3 (chick, >384 amino acids; Gould et aI 1992) syndecan2 (human, 20 1 amino acids; Marynen et aI1989), and syndecan-4 (rat, 202 amino acids; Kojima et aI1992b). Each protein contains a cluster of similar putative GAG attachment sites distal from the plasma membrane near the N-terminus of the mature protein: one site (or two in syndecan-3) is of the syndecan-type sequence and the other two (or three) sites are of the serglycin-type. An SA site is included for syndecan-2, as previously suggested (Marynen et al 1989). Syndecan-l and -3 contain putative GAG attachment sites that are absent in syndecan-2 and -4. The proposed protease susceptible site adjacent to the transmembrane domain is highly conserved. The se quences of the C-terminal region of the rat proteins are shown in Figure 3.
membrane, are both syndecan-type in sequence , and are e ncoded by separate exons . The more N-terminal of these sites is encoded by a relatively large exon in the rat and chick syndecan-3 gene , which in part resembles the T- , So, and P-rich regions in mucin-like proteins . These sites are not uniformly substituted on syndecan-l from mouse mammary epithelial (NMuMG) cells . Most of the molecules contain only chondroitin sulfate chains at these s ites , but a minor but significant proportion contains both chondroitin sulfate and HS chains (R. Kokenyesi , unpublished). The regions C-terminal to the conserved putative protease-susceptible site are most highly conserved (Figure 3). A single exon in the mouse syndecan- l
374
BERNFIELD ET AL
Syndecan-1
L
Y
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Syndecan-3 Syndecan-2
K RfT]E V L A A V I A G G V[DG F L F A[DF LrJL L L V Y
Syndecan-4
E Rl.:JE
v
Syndecan-1
R M KKK
Syndecan-3
R M KKK D E G S Y T
L A A L I�G G
v v
G I L F AV F
L
W
L
L LV Y
F YA
D E G
Syndecan-2 Syndecan-4
Figure 3
R M KKK
DE G S Y D L G -
Derived amino acid sequence of the highly conserved C-terminal regions of the rat
syndecan core proteins, which show the region containing the putative protease susceptible site, the transmembrane domain, and the cytoplasmic domain (Cizmeci-Smith et al 1992; Pierce et al 1992; Carey et al 1992; Kojima et al 1992b). The amino acids conserved in the sequences are
maximal identity, syndecans-I and -3 (70%) and syndecans-2 and -4 (77%) are adjacent to each other. The putative protease sites (bold) eontain two basic residues in syndecans-I, -2 anmd -3, but solely arginine in syndecan-4. The transmembrane domains and the stop transfer sequence are of the same length in each protein, within the box in the diagram; the sequences showing
while the remainder of the cytoplasmic domains ean be aligned only by introducing several
independent deletions. A tyrosine (bold) at the end of the transmembrane domain and three tyrosines (bold) in the cytoplasmic domain are conserved in each protein. The loop between the first and the second tyrosin e of the cytoplasmic domain is the most disparate region of this domain
and varies in sequence and in length. This region and the loop between the second and third tyrosine of syndecan - l and -3 are quite similar, as are these regions in syndecan-2 and -4.
gene and in the rat and chick syndecan-3 genes encodes the identical portion of this region. The tyrosine that completes the transmembrane domain and the three tyrosines in the cytoplasmic domain are invariant. The length and sequence between the transmembrane domain and the first tyrosine are con served and could account, in syndecan-l and -3, for a tyrosine internalization signal (Ktistakis et al 1990). However, the distance between the next tyro sines differs,
possibly providing individual syndecans with
specificity
towards interacting proteins. One of the tyrosines fits a consensus sequence for tyrosine phosphorylation (Gould et al 1992; Pierce et al 1992). Comparisons between species of derived protein sequences for the syn decans show an intriguing evolutionary relationship (Figure 4). Syndecan-l sequences of the mouse (Saunders et al 1989a), human (Mali et al 1990), hamster (Kiefer et al 1990), and rat proteins (Cizmeci-Smith et al 1992;
THE SYNDECANS
100%
Rat
l?dl
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I� I II
88%
III I I 1111111 II II
@
I
1111111 11I1II
82%
III lUI II I
���y
III
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yyyy
� Signal Sequence m Transmembrane
I
96% 100%
96% 100%
70%
91%
Human
I I
If'�1!X/Ad ���� I II I I f?jll 111111111111111111111111111111 11 [ 111�/:}\:: '.: 1
95%
Hamster
II
II
Mouse
375
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flliJ Cytoplasmic
o
Extracellular
Potential sites:
I
GAG Attachment
1 Protease Cleavage
Figure 4 Diagram showing the core protein domain structure and sequence conservation of syndecan-I derived from the mouse (Saunders et al 1989a), rat (Cizmeci-Smith et al 1992; Kojima et al 1992b), hamster (Kiefer et al 1990), and human (Mali et al 1990) cDNAs. Percent identity to the mouse protein is shown above each domain and the black lines within a domain I (bar) indicate the position of the amino acids that differ from the mouse protein sequence. Lines above the bars indicate the putative glycosaminoglycan attachment sites. The locations of the invariant tyrosines are also shown.
Kojima et al 1 992b) show that the cytoplasmic domain and , not unexpectedly, the transmembrane domain do not evolve among these species, but the extracellular domain is evolving extremely rapidly, except for highly con
served sequences surrounding the GAG attachment sites and at the putative A nearly identical pattern of conservation is seen in the mouse , human , and rat syndecan-2 proteins. The extracellular domains of human and mouse syndecan-1 show only 70% sequence identity and thus these domains diverge as rapidly as plasma albumin and lysozyme , proteins with very rapidly evolving protein sequences (Doolittle 1992) . Moreover , the available rat and chick syndecan- 3 and rat and chick syndecan-4 sequences are 57 and 52% identical, respectively, which ranks them among the fastest changing vertebrate proteins (Doolittle 1 992) . The sequence comparisons suggest that the functional elements of the extracellular domain are the GAG chains and the protease susceptible sites near the plasma membrane . Moreover, the cytoplasmic domain likely inprotease-susceptible site.
376
B ERNFIELD ET AL
teracts with evolutionarily stable components such as cytoskeletal (e.g. actin, tubulins) and regulatory (e.g. calmodulin) proteins. Distinct evolutionary stability of extracellular and cytoplasmic domains is also seen in the tyrosine kinase and tyrosine phosphatase families of membrane-spanning proteins (Doolittle 1991). These, like syndecan-l , function as receptors at the cell surface.
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FUNCTIONS OF THE SYNDECANS Matrix Receptor All adherent cells detect and respond to the extracellular matrix, an insoluble amalgam of large multidomain proteins. The matrix produced by mesenchy mal cells accumulates in the interstitial spaces, whereas the matrix produced by epithelial cells organizes into a basement membrane (for review, see Bemfield et aI1984). Syndecan-l binds cells via its HS chains to a variety of components of the interstitial matrix, including types I, III, and V collagen, the fibrillar collagens (Koda & Bemfield 1984), fibronectin (Saunders & Bemfield 1988), thrombospondin (Sun et al 1989), and tenascin (Salmivirta et al 1991). This binding is independent of calcium and magnesium ions (Koda et al1985; Saunders & Bemfield1988) and is abolished by trypsin treatment of cells, thus distinguishing it from matrix binding via the integrins, a family of heterodimeric proteins receptors (Albelda & Buck 1990). At physiological pH and ionic strength, similar binding is not seen with the protein-free GAG chains isolated from syndecan-l of NMuMG cells. Under these conditions, the PG binds with relatively high affinity (Kd for type I collagen fibrils is ca. 1 nM) (Koda & Bemfield 1984), but binds to neither laminin nor type IV collagen, both components of the epithelial basement membrane (Koda et al 1985; Elenius et al 1990). Binding to collagens appears to require the fibril; stochiometry is 2-300 type 1 collagen monomers per each syndecan-l mole cule, varying with the fibril preparation, and no binding is seen to denatured collagen or to individual collagen monomers (Koda & Bemfield 1984; Koda et al 1985). Although these binding properties alone are not sufficient to implicate syndecan-l as a physiologically relevant matrix receptor, its expression is also consistent with this role. It polarizes to the basolateral surfaces of simple epithelia, both in culture (Rapraeger et al 1987) and in tissues (Hayashi et al 1987), and localizes in early mouse embryos to the site of initial matrix accumulation (Sutherland et al 1991). Syndecan-l binds cells of the B-cell lineage to type I collagen (Sanderson et al 1992b), and its expression corre lates with the sites and times that B cells associate with matrix (Sanderson et al 1989): It is expressed on precursor B cells in the bone marrow, but not after the cells mature and are released into the circulation as lymphocytes. Syn-
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THE SYNDECANS
377
decan- l reappears when the cells differentiate into plasma cells in lymphoid tissues. Other properties also implicate cell surlace syndecan-l as a matrix recep tor. It is shed within minutes from polarized NMuMG cells when they are suspended and is not replaced on the cell surlace until these cells re-attach to a substratum (Jalkanen et al 1987 ) . While most syndecan-l in non-polarized, mammary epithelia is detergent-extractable, which suggests that it is free in the membrane, cross-linking of cell surlace syndecan- l by antibodies or matrix molecules induces it to associate with the actin-containing cytoskele ton and resist detergent extraction (Rapraeger et aI 1 987) . Indeed, syndecan- l and F-actin filaments co-localize at basal cell surlaces in polarized mammary epithelia (Rapraeger et al 1 987; Kato & Bemfield 1 990), and isolated syn decan- l , but not its extracellular domain, binds directly or indirectly to F-actin filaments in a sedimentation assay (Rapraeger & Bemfield 1 982) . Thus, syndecan-l may link the matrix to the cytoskeleton. These results are most consistent with syndecan- l acting as a matrix receptor, at least for epithelia where it is predominantly found in mature organisms. Because of its high abundance (ca. 1 06 syndecan- l molecules per NMuMG cell surlace; M. Bemfield, unpublished) and its relatively high binding affinity, it is potentially important that syndecan-l is cleaved from the plasma membrane when epithelial cells are induced to change shape. This mechanism enables rapid and possibly controlled release of the cells from the matrix, which accommodates cell movements or changes in cell shape during morphogenesis and tissue repair . Although its extended conformation might enable the HS chains to interact, its binding preference for interstitial matrix rather than basement membrane components is inconsistent with this role for epithelia. Syndecan- l also localizes to cell surlaces where there is no matrix apparent , e.g. at the lateral surlaces of simple epithelia and over the entire surface of stratified epithelia, and is markedly diminished in amount and not concentrated at adhesive sites on mesenchymal cell surlaces (Kato & Bem field 1 989) . These distributions suggest additional functions for syndecan- l . Organization of Epithelia
Syndecan-l expression correlates with normal epithelial cell organization in vitro. Studies with a mouse mammary tumor cell line (S 1 15 cells) that lose an epithelioid morphology and become fusiform in response to steroid hormones (Yates & King 1 98 1 ) i mplicate syndecan- l in organizing epithelial cells into sheets (Leppa et al 1 99 1 ) . When these cells are induced to change shape by testosterone, the levels of cell surlace syndecan- l and mRNA fall. Glucocor ticoids also cause these cells to become fusiform. When these cells are transfected with a syndecan- l cDNA under the control of a glucocorticoid responsive MMTV-LTR, hormone-induced expression of syndecan- l pre vents the change in morphology. While the parental S115 cells, similar to
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BERNFIELD ET AL
other carcinoma cells, grow on a soft agar substratum, the hormone-treated transfectants fail to grow, which suggests that syndecan- l expression restores at least one aspect of the non-transformed phenotype (Leppii et al 1992) . NMuMG cells made syndecan - l-deficient by stable transfection with the syndecan- 1 coding region in an antisense configuration show two distinct morphologies: clones bearing more than 45% normal levels of cell surface syndecan- l grow as islands of closely adherent epithelia, whereas clones showing less than lO% normal levels grow as individual fusiform cells that overlap each other (Saunders et al 1 989b; Kato & Bemfield 1 990) . Whereas vector-only controls grow as sheets on top of collagen gels and as duct-like structures within it, the fusiform cells invade the gels and migrate within them, mimicking the behavior of authentic mesenchymal cells. The fusiform cells retain the milk fat globule antigen and cytokeratins, and thus have mammary epithelial characteristics, but lose an epithelia-like organized actin cytoskeleton, redistribute 13 1 integrins , and markedly decrease E-cadherin expression. The cytoskeletal, 13 1 integrin , and E-cadherin changes in syndecan- 1 deficient cells may be a consequence of the change i n cell shape resulting from loss of a matrix receptor. However , syndecan- l co-localizes with F-actin bundles at the lateral surfaces of NMuMG cells (Rapraeger et al 1987). The se bundles are apparently associated with the zonula adherens, the cellular organelle responsible for epithelial organization. Certain 13 1 integrins and E-cadherin, the major epithelial cell adhesion molecule (Takeichi 1 99 1 ) , are also found at this site . Thus, syndecan- l may participate in a membrane cytoskeletal complex of adhesion proteins that maintains the organization of epithelial sheets . Such a complex may exist at various sites within epithelial cells, which enables syndecan- 1 to be a member of a matrix receptor complex at the basal cell surface and an adhesion complex at adherens junctions. A role for syndecan- 1 in maintaining epithelial organization is also sug gested by its expression during epithelial-mesenchymal transformations in embryos . During development of the secondary palate in rodents, the medial edge epithelia of the nasal and maxillary processes transform into mesen chymal cells (Fitchett et al 1 990). Immediately prior to this transformation and precisely at the sites where the epithelia will become fusiform cells, syndecan- 1 expression is markedly reduced , and a decrease in E-cadherin expression follows (Fitchett et al 1 990, K . McAlmon, unpublished) . The intermediate filaments then switch from cytokeratins to vimentin and these now fusiform cells migrate into the interstitium. Non-thrombogenic Vascular Endothelial Cell Surfaces
Heparin has long been used clinically as an anticoagulant . The heparin-like GAGs promote anticoagulation by interacting with the proteases of the in trinsic coagulation cascade and thrombin, as well as a variety of inhibitors,
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THE SYNDECANS
379
including protein C and the serpins, a family of serine protease inhibitors that includes antithrombin Ill . A specific pentasaccharide sequence found in heparin and HS binds to antithrombin III, thereby enabling it to form an enzymatically inactive complex with several coagulation enzymes (Daniels son et al 1986) . A similar interaction with protease nexins promotes the inactivation of pericellular proteases (Farrell & Cunningham 1986) . Anticoagulantly active HS on vascular endothelial cell surfaces could endow them with non-thrombogenic properties (Colburn & Buonassisi 1 982). Rat fat pad microvascular endothelial cells produce such HS chains, which accounts for less than 5% of their total HSPG (Kojima et al 1992a). While a low proportion , this is five- to tenfold more anticoagulant HS than produced by bovine aortic endothelial cells (Marcum et aI 1 986) . Importantly, these HS chains are not on a single type of core protein , but are on syndecan- l and -4 and possibly other cell surface PGs (Kojima et al 1992a). On average, each anticoagulantly active HS chain contains a single interacting site. Therefore, at least in these cells , the primary sequences of the different syndecan core proteins do not dictate enrichment of their HS chains for the distinct anti thrombin III binding structure. These microvascular endothelial cells contain more syndecan- l than syn decan-4 mRNA and protein , while rat aortic smooth muscle cells and rat fat pad adipocytes (Kojima et al 1992b) contain more syndecan-4 mRNA . Syn decan- l in bovine pulmonary artery endothelial cells is primarily an in tracellular molecule, as in other mesenchymal cells . The small proportion on the cell surface localizes predominantly at the basal surface , the putative abluminal surface (Lose & Bernfield 199 1 ) . Thus, other HSPGs may also be involved in producing a non-thrombogenic luminal surface. Partner in bFGF Action
Fibroblast growth factors (FGF) are a family of heparin-binding growth factors that promote the proliferation and migration of mesenchymal cells and the survival and differentiation of neural cells . Currently, there are seven members of this family, and these have a variety of sources and biological roles , including angiogenesis , embryonic inductions , repair of injury, and neoplastic transformation (see Burgess & Maciag 1989 for review) . Cell surface HSPGs are apparently required for the bFGF action and may be involved in the action of other members of the FGF family (Klagsbrun & Baird 199 1 ) . The binding of bFGF to heparin and to HSPG in the basement membrane and at the cell surface is thought to be involved in bFGF activity because mutation of the heparin/HS binding site on bFGF reduces its biologi cal activity (Seno et al 1 990) . The activity of acidic FGF is also stabilized by heparin and, at least in a parathyroid cell line, requires a cell surface HSPG (Sakaguchi et al 199 1 ) . Cell surface H S appears to be required for the binding of bFGF to its high
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BERNFIELD ET AL
affinity receptor . CHO cells defective in HS synthesis do not bind bFGF even after inducing the expression of FGFR- l , a signal-transducing high affinity bFGF receptor . They will bind, however , when exogenous heparin is avail able, which suggests that binding to cell surface HS is a prerequisite for high affinity binding (Yayon et al 1 99 1 ) . This work was extended by findings that cells made deficient in cell surface HS , by heparitinase pretreatment , or by chlorate treatment to prevent sulfation, have a markedly decreased ability to respond to bFGF as assessed by proliferation (Swiss 3T3 cells), or by repression of terminal differentiation (MM14 myoblasts) (Rapraeger et al 1 99 1 ) . These studies do not identify a specific cell surface HSPG as the low affinity receptor for bFGF but syndecans are prime candidates. Syndecan- l from mammary epithelial cells binds bFGF via its HS chains (Bem field & Hooper 1 991), and cell surface expression of hamster syndecan-l in l ympho blastoid cells endows these cells with low-affinity binding sites for bFGF (Kiefer et al 1 990) . Moreover , expression of syndecan- l correlates with the potential activities of bFGF or its family members during development (see section on developmental regulation). Syndecan- l expression during early mouse development corresponds with the sites of bFGF action in inducing axial mesoderm formation in Xenopus laevis (Amaya et al 1 99 l ) . Syndecan- l is also expressed transiently on a variety of condensing mesenchymal cell popUlations (e. g . tooth , kidney, limb) when they are rapidly proliferating and is lost when these cells differentiate, consistent with the known effects of bFGF on mesenchymal cells . Syndecans as Co-receptors
Several functions proposed for the syndecans involve binding components of the cellular microenvironment that themselves interact with a cell surface receptor (Figure 5). For example, fibronectin and interstitial collagens bind syndecan- l , but also bind various integrin receptors, and bFGF binds syn decan- l, but also associates with its high affinity protein receptor. Many additional interactions are possible because heparin and HS bind a broad range of matrix components , growth factors , cell adhesion molecules, degra dative enzymes, and protease inhibitors (cf Table 1 ) . For instance , heparin binding EGF also binds the EGF receptor (Higashiyama et al 1 99 1 ), N-CAM also has homophilic interactions (Reyes et al 1 990), and thrombin also interacts with a specific receptor (Pouyssegur & Seuwen 1 992) . The signifi cance of these dual interactions is unclear, but binding to cell surface HS is often not suspected because it is ubiquitous. Thus, cell surface HSPGs may be co-receptors, integral membrane components that are structurally distinct from high affinity receptors , which bind biological ligands and participate in formation of a receptor complex that is required to generate the ligand's physiological action.
381
THE SYNDECANS
Syndecans
+
F.
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