Lysosomal enzyme targeting STUART KORNFELD Department of Medicine, Division of Haematology-Oncoloa, Washington University School of Medicine, Mi0 South Euclid, P.O. Box 8125. St Louis, MO 63110, U.S.A.

Historical overview I want to express my appreciation to the Biochemical Society for having invited me to deliver the 1989 Jubilee Lecture. It is truly a great honour. The word Jubilee connotes a very special anniversary, and in this spirit I want to impart an historical overview to the Lecture in addition to reviewing my most recent work. This is especially appropriate because my work on lysosomal enzyme targeting has built on the remarkable achievements of previous investigators in this field. It is almost 40 years since the classic studies of Christian DeDuve and his colleagues resulted in the discovery of lysosomes as a special group of cytoplasmic particles that contained numerous acid-dependent hydrolases [ 11. This early work is described in Dr DeDuve’s Jubilee Lecture given in 1978 [ 2 ] . After these seminal observations, there was a flurry of studies by DeDuve, Straus, Novikoff, Cohn, Tappel and others which established that lysosomes are responsible for degrading both internalized and endogenous macromolecules. These early studies were summarized in a wonderful Ciba Foundation Symposium which was published in 1963 [3].That same year Hers reported the first case of a lysosomal storage disease, thereby initiating a new chapter in the study of this organelle [4].At the present time more than 40 different lysosomal storage diseases have been described. Most of these disorders are characterized by a deficiency of a single lysosomal enzyme which results in a massive accumulation of the material that is normally degraded by that enzyme. It was studies of cultured fibroblasts from patients with genetic disorders of mucopolysaccharide catabolism that initiated the next phase of research in this field, which was the investigation of the biogenesis of lysosomes. In a series of insightful experiments, Elizabeth Neufeld and her colleagues observed that normal fibroblasts secrete ‘corrective factors’ that are rapidly endocytosed by enzyme-deficient cells, resulting in the degradation of the accumulated mucopolysaccharides [S]. When these corrective factors were purified, they were found to be the particular lysosomal enzymes that were missing in the various mutant fibroblasts. Neufeld also noted that the lysosomal enzymes were taken up in a selective and saturable manner, indicating that they contained a recognition marker that allowed high-affinity binding to receptors on the fibroblast surface. Evidence for the presence of a common recognition marker came from studies of patients with I-cell disease (mucolipidosis ll), a rare autosomal recessive disorder characterized by the intracellular deficiency of many lysosomal enzymes in the face of elevated levels of these enzymes in the patient’s plasma. Hickman & Neufeld observed that fibroblasts from I-cell patients take up and retain normal lysosomal enzymes in the usual fashion, whereas similar hydrolases secreted by I-cell fibroblasts are not taken up by normal fibroblasts [6]. This led the authors to propose that the enzymes secreted by the I-cell fibroblasts lack a recognition marker required for adsorptive pinocytosis. Subsequent studies indicated that the oligosaccharides on acid hydrolases were involved in the recognition by the cell surface receptors [7, 81, but the identity of the functional group remained obscure until Kaplan et al. demonstrated that the adsorptive pinocytosis of VOl.

18

Jubilee Lecture Delivered on 19 December 1989 at St Bartholomew’s Hospital Medical College, London

PROFESSOR S. KORNFELD

lysosomal hydrolases could be specifically inhibited by mannose 6-phosphate (Man6P) [9]. Sly and his co-workers went on to demonstrate that lysosomal enzymes contained Man6P and that their uptake was proportional to the Man6P context of the enzyme [lo].Thus, Man6P was proven to be the critical component of the common recognition marker whereby lysosomal enzymes are bound and internalized by fibroblasts; the receptor that binds the phosphorylated lysosoma1 enzymes was subsequently referred to as the Man6P receptor (MPR). The presence of cell surface receptors for lysosomal enzymes, together with the secretion of lysosomal enzymes capable of binding to these receptors, led to the suggestion that lysosomal enzymes are normally secreted and then recaptured for packaging into lysosomes [ 71. However, 367

BIOCHEMICAL SOCIETY TRANSACTIONS

368 it was subsequently shown that this pathway accounts for the delivery of only a small proportion of newly synthesized lysosomal enzymes to lysosomes, with the major routeing being intracellular [ 1 1- 13). While these studies established the biological importance of the phosphomannosyl recognition system, a key component that was missing was the mechanism by which the mannose residues of the lysosomal enzymes are phosphorylated. Therefore, in 1979, Ira Tabas, a M.D./Ph.D. student and I decided to study this problem. Ira had just completed a series of studies demonstrating that the asparagine-linked oligosaccharides on newly synthesized glycoproteins are processed from high-mannose to complex-type units [ 141. In these experiments, we used [2-3H]mannoseto label the oligosaccharides since it had been established that the initial glycosylation of the asparagine residues occurred by the en bloc transfer of a pre-formed high-mannose-type oligosaccharide from a lipid carrier to the nascent polypeptide chain. Therefore, we reasoned that with the [2--Hlmannose label we could follow the fate of the oligosaccharide units of newly synthesized lysosomal enzymes as they proceeded from their site of synthesis to their final destination. This would allow us to determine the point at which phosphorylation occurs. The availability of [3H]mannose-labelled oligosaccharides would also facilitate any subsequent structural studies. Using this approach, we were able to show that newly synthesized /3-glucuronidase initially acquired phosphate in the form of a diester between the hydroxyl group on C-6 of a mannose residue and C-1 of an outer a-linked Nacetylglucosamine [ 151. Ajit Varki then carried out an extensive structural analysis on the phosphorylated oligosaccharides to establish the structures shown in Fig. 1 [ 161. We found that all of the phosphomannosyl residues were present on high-mannose-type



or hybrid-type molecules, and that the oligosaccharides contained either one or two phosphates located on five different mannoses, as denoted by the asterisks in Fig. I . In addition to the phosphodiesters, some of the oligosaccharides contained phosphomonoesters, and subsequent pulse-chase experiments showed that the diesters were the precursors of the monoesters [ 171. This same phosphodiester linkage was described simultaneously by Hasilik et ul. in human fibroblast /3-hexosaminidase [ 181. The most important implication of the finding of the phosphodiester-linked moieties was that it suggested a possible enzymic pathway for the phosphorylation of lysosomal enzymes (Fig. 2). We proposed that the phosphomannosyl residues were formed by a two-step reaction. First, a-Nacetylglucosamine 1-phosphate would be transferred from UDP- N-acetylglucosamine to a mannose residue of a lysosoma1 enzyme high-mannose unit. Then the blocking moiety would be removed to generate the phosphomonoester recognition marker. This scheme predicted the presence of two novel enzymes and my laboratory, as well as the laboratory of Kurt von Figura, soon demonstrated the existence of both enzymes in a number of different tissue cell types [ 19-22]. Our laboratories then showed that the first enzyme in the series, UDP-GlcNAc: lysosomal enzyme N-acctylglucosamine-1-phosphotransferase is defective in patients with I-cell disease and the related disorder, pseudo-Hurler polydystrophy (ML Ill) (21, 23, 24). This accounted for their failure to generate the phosphomannosyl recognition marker on their lysosomal enzymes. Specifciy of lysosomul enzyme phosphotylutiori

One of the most intringuing questions about this targeting system concerns the basis for the specificity of the selective phosphorylation of oligosaccharide units on lysosomal enzymes. Since structural studies indicated that lysosomal enzymes and non-lysosomal glycoproteins are both glycosylated with the identical oligosaccharide precursor, it seemed likely that the lysosomal enzymes would contain a common protein domain that is recognized by phosphotransferase. Using a partially purified preparation of rat phosphotransferase, we showed that lysosomal enzymes are much better acceptors for phosphotransferase than non-lysosomal glycoproteins (Fig. 3) [25]. The apparent K,,, values for lysosomal enzymes were in the low micromolar range, whereas the K, values for non-lysosomal glycoproteins were at least 100-fold greater. The enzyme could also act on the carbohydrate acceptors, a-methylmannoside and Man,_,GlcNAc oligosaccharides, but these molecules had very high apparent K, values, some 103-10J-fold greater than those of lysosomal enzymes. Taken together, these data indicated that the high-affinity interaction between the phosphotransferase and lysosomal enzymes was mediated

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primarily hy protein-protein interactions. This wiis shown directly hy demonstrating that dcglycosylntcd lysosomal eiuynics arc potent inhibitors of the phosphorylation of intact lysosonial enzymes 1201. T h e specificity of the phosphotr;insfcrase towards lysosomnl enzymes made it likely that it was the initial and determining enzyme in the pathway which eventually results i n thc segregation o f acid hydrolases into lysosomcs. Further insight into the nature o f the phosphorylation process has conic from studies o f ;I suhset o f the patients with pseudo-tiurler polydystrophy 124. 271. T h e phosphotransfcrnsc o f these patients' fibroblasts has normal activity towards a-niethylmannoside, ;I substrate that is phosphorylatcd independent o f protein recognition. hut poor activity towards lysosomal enzyme acceptors. T h e mutant cnlymc. therefore. is defective in its protein recognition function. These dnta led to the proposal that phosphotransferase is iin enzyme that contains ;I recognition site (or subunit) and ;I catalytic site (or subunit) that interact to specificnlly recognize and phosphorylate lysosomal enzymes. T h e defect in the mutant phosphotransferase would be in the recognition site (Fig. 4). T h e actual identity of the common protein recognition domain of lysosomal enzymes has heen difficult t o elucidate for several reasons. First, it is not ;I simple linear sequence o f amino acids. since the numerous lysosonial enzymes that have hccn cloned d o not share any significant sequence identity. Furthermore. since heat-denatured lysosomal enzymes o r proteolytic fragments of lysosomal enzymes d o not serve a s substrates o f the phosphorylating enzymc. it appears that the conformation o f the protein is important for the expression o f the recognition marker 125 1. Recently. some insight into the nature o f the recognition domain has come from studies o f chimeric proteins derived from two aspartyl proteascs, pepsinogen and cathcpsin D (T. Baranski. P. F'aust & S. Kornfeld. iinpublished work). Although these two proteins share 50% identity in amino acid sequence. pcpsinogen is a secretory protein, whereas cathepsin D is a lysosomal enzyme. When pepsinogen. engineered t o contain sites for Asn-linked glycosylation at the same positions a s cathcpsin D. was expressed in Xer~opirsoocytes, it was glycosylated and secreted. but not phosphorylated. Cathepsin 0. VOl. 18

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Fig. 4. ,\lodcljOr l\:so.sor~itrl C I I : \ ~ I ~ ~t ~ ~ ~ o g t i i t iI)), o t iN-trcc/yl~~Iirc~o.str~~ri~iyl~~lio.s~~liot~~trri.s~rir.se Enzyme from normal individuals. ( h ) Enzyme from patients with a variant form of pseudo-t turler polydystrophy (ML-Ill). T h c defect in these patients' enzyme is postulated t o he in the protein-recognizing function rather than i n the catalytic site where mnnnose phosphorylation occurs. T h e on the lysosomal enzyme represents the proposed protein domain recognized by the phosphotransfer~ise.m. NAcetylglucosamine; O , mannose; Urd, uridine.

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on the other hand, wah efficiently phosphorylnted when expressed in the oocytes 1281. Phosphorylation also occurred using chimeric proteins containing only two regions o f cathcpsin D (residues 188-230 and 205-3 19) spliccd into the pcpsinogen backbone. Chimeras containing either o f these regions alone were not phosphorylnted. When the positions of these residues are localized on the three-dimcnsional model o f pepsin 12Yl. it is apparent that they ;ire in close apposition t o each other on the surface o f the molecule. T h e implication is that the recognition marker is formed by residues derived from different regions o f cathcpsin I). This conformational nature o f the recognition domain would explain the failure to detect amino acid seqiiencc identity between lysosomal enzymes.

370

BIOCHEMICAL SOCIETY TRANSACTIONS

M W recept0r.s The next step in the targeting of newly synthesized lysosoma1 enzymes is their binding to MPRs in the Golgi. As previously noted, the initial binding studies with purified ‘corrective factors’ indicated the presence of receptors with high affinity for lysosomal enzymes containing phosphomannosyl residues. In 1981, Sahagian et al. reported the isolation of a membrane-associated glycoprotein with an apparent M , of 215 000 that bound lysosomal enzymes with high affinity 1301. Ligand binding to this receptor was independent of divalent cations. While the 2 15 000-M, MPR was shown to be widely distributed, we subsequently identified a number of murine tissue culture cell lines that were deficient in this receptor (311. In spite of having no detectable or barely detectable receptor, the cells still sorted 30-40%, of their newly synthesized lysosomal enzymes to lysosomes, indicating the presence of a targeting mechanism that was independent of the 215 000-M, MPR. Since the sorting of lysosomal enzymes from secretory proteins in these cells seemed likely to be receptor-mediated, Bernard Hoflack and I decided to look for conditions whereby lysosomal enzymes would bind to the membranes of these cells. We found that lysosomal enzyme binding would occur when divalent cations were present, and that the binding was saturable and behaved like a receptor-mediated process [32]. However, to our great surprise, the binding was completely inhibited by Man6P, indicating the presence of a second MPR that participates in the delivery of acid hydrolases to lysosomes. This receptor was then purified and shown to be an integral membrane glycoprotein with a subunit apparent M , of 46 000 [33]. On the basis of their differences in divalent cation requirements, the large receptor was referred to as the cation-independent (C1)MPR and the small receptor as the cation-dependent (CD)MPR. Some of the properties of the receptors are summarized in Table 1. Both receptors have similar, but not identical, binding specificities towards various types of phosphorylated oligosaccharides, preferring oligosaccharides containing phosphomonoesters over those with phosphodiesters (341. However, the CIMPR, but not the CDMPR, will bind a phosphomannosyl residue diesterified t o a methyl group, as occurs in the lysosomal enzymes of Dictyostelium discoideum [35]. The best ligand for both receptors is an oligosaccharide containing two phosphomonoesters. Equilibrium dialysis studies have revealed that the CDMPR binds 1 mol of Man6P or 0.5 mol of the divalent phosphorylated oligosaccharide per monomer [36]. Since chemical cross-linking experiments indicate that the CDMPR is a dimer in the membrane (37, 381, each Table 1. I’roperties ofthe t w o Mmhl’ recept0r.s

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Yes 275 000 ‘? Monomer Man6I’ Man6I’-OCH, IGF-11 No

Yes 46 000 Dimer Man6I’

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Lysosomal enzyme targeting.

Lysosomal enzyme targeting STUART KORNFELD Department of Medicine, Division of Haematology-Oncoloa, Washington University School of Medicine, Mi0 Sout...
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