151
J. Anat. (1990), 170, pp. 151-160 With 6 figures Printed in Great Britain
N-linked oligosaccharides during human renal organogenesis S. FLEMING*
University Department of Pathology, Southampton General Hospital, Southampton S09 4XY
(Accepted 18 December 1989) INTRODUCTION
There is interest in the developmental stage specific expression of cell surface carbohydrates which may have a function in cell interactions during embryogenesis. The cell surface expression of fucosylated derivatives of poly-N-acetyl lactosamine has particularly been shown to be important during mammalian development (Feizi & Childs, 1985). These glycoconjugates have been shown to be expressed in a characteristic distribution (Solter & Knowles, 1979), and to be involved in cell interactions at the morula stage and during gastrulation of pre-implantation mammalian embryos (Jacob, 1979). Related molecules are expressed on the cell surface of some cells during the differentiation of fetal tissues (Kelly & Fleming, 1987). Fleming & Brown (1986) have recently shown that fucosylated derivatives of N-acetyl lactosamine are expressed at important sites of organogenetic interactions during the development of the human kidney. Demonstration of these cell surface molecules has been achieved by utilising the specificity of monoclonal antibodies (Lloyd, 1987). The monoclonal antibodies used in these investigations are specific for the terminal oligosaccharide epitopes which are carried on larger carbohydrate side chains of glycolipids or glycoproteins (Feizi, 1984). Although these molecules can be carried on either lipids or proteins, Ozawa, Muramatsu & Solter (1985) have shown that during important stages of embryogenesis the saccharides are frequently carried on a large molecular weight glycoprotein which they have called embryoglycan. It is now possible to analyse parts of the structure of the remainder of the carbohydrate side chains and their linkages to peptides by the use of specific glycosyl hydrolases (Rastan et al. 1985). These are enzymes, mostly of plant or bacterial origin, which hydrolyse linkages of a specific configuration between defined saccharides. Thus, by testing the sensitivity of monoclonal antibody binding in immunocytochemical experiments to pre-digestion by specific glycosyl hydrolases, the structure of the carbohydrate side chain carrying the appropriate epitopes may be inferred. We have studied the distribution of fucosylated derivatives of the Type II blood group substance during the development and differentiation of the human kidney using monoclonal antibodies and the immunoperoxidase technique. We have further analysed the structure of these glycoconjugates by using glycosyl hydrolases.
* Present address: Department of Pathology, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, Scotland.
152
S. FLEMING
LeX
P-Gal 1-4 GLCNAC 3
FUC
H-type 11
«-Gal 1-4 GLCNAC 2
FUC Fig. 1. The structure of the oligosaccharides conferring LeX and H-Type II antigenicity. MATERIALS AND METHODS
Tissues Both cryostat-sectioned and formalin-fixed, paraffin-embedded tissues were used. Similar results were obtained with both types of tissue. Fetal kidneys were dissected from fetuses of gestational ages between 12 and 18 weeks which were obtained during legal terminations of pregnancy. For cryostat sectioning a bisected fetal kidney was snap-frozen in liquid nitrogen and stored at -70°C until use. Six micron thick cryostat sections were cut, one section of which was stained with haematoxylin and eosin. Multiple adjacent cells were used for immunocytochemical experiments after brief fixation in cold acetone. From each fetus the second kidney was fixed in 10% neutral buffered formalin for 24 hours. The tissues were then processed to paraffin wax and 4 ,um sections cut. They were stained with haematoxylin and eosin and multiple adjacent sections were used in immunocytochemical experiments. Ten specimens of each type of tissue were used in these experiments. Antibodies The murine monoclonal antibodies, AGF4-48 and Leu-M 1, are both reactive against the LeX hapten. The structure of this antigenic determinant is shown in Figure 1. This oligosaccharide is also responsible for the reactivity of the antibody, SSEA-1, and the human leucocyte marker, CD15 (Gooi et al. 1981). Both AGF4-48 and LeuMl show CD 15-like immunoreactivity in their tissue distribution and their specificity for the above oligosaccharide has been confirmed by competitive inhibition of binding, using synthetic oligosaccharides (Kelly & Fleming, 1987). A commercially available (DAKO) antibody to human blood group Substance H on a Type II precursor chain was also used. The epitope recognised by this antibody is illustrated in Figure 1. All three of these antibodies are murine IgM molecules.
Glycosyl hydrolases Several glycosyl hydrolases were used. Sections were incubated with these enzymes before incubation with the primary antibodies in the immunocytochemical technique. Neuraminidase (EC 3.2. 1 .18) from Clostridium perfringens removed terminal neuraminic acid from carbohydrate chains. Tissue sections were incubated with neuraminidase for 2 hours at room temperature and at pH 4-6. The structure of the poly-N-acetyl lactosamine carbohydrate backbone of the
Oligosaccharides and renal organogenesis [21
Fucosidase
NEU
FUC ± GAL
GAL
.
GLCNAC I GAL
GLCNAC 4GAL
-
153
Neuroaminidase
Endogalactosidase
l
GLCNAC
GLCNAC
l MAN
MAN
MAN
GLCNAC
4-
Endoglycosidase F
GLCNAC %
Glycopeptidase F
ASN Fig. 2. The cleavage sites on N-linked oligosaccharides of the endoglycosidases.
antigens can be inferred from the sensitivity to digestion by endo-beta-galactosidase (Scudder, Horfland, Ibemura & Feizi, 1984). This enzyme will digest linear chains of galactose and N-acetyl-glucosamine but will not digest branched chains of the same carbohydrates. It, therefore, distinguishes between the human blood group antigen, i, which is enzyme-sensitive, and blood group, I, which is enzyme-resistant. Endo-betagalactosidase (EC 3.2.1.103) purified from Bacterioides fragilis by the method of Scudder et al. (1984) was incubated with tissue sections at an activity of 0 1 U per ml and at pH 5-8. N-linked oligosaccharides are sensitive to digestion by glycopeptidase-F. This enzyme cleaves high mannose hybrid and complex glycans from glycoproteins between the ASN of the peptide and the GLCNAC of the carbohydrate (Plummer et al. 1984). The glycopeptidase-F (EC 3.2.2.18) used in these experiments was purified from Flavobacterium meningosepticum and was used at an activity of 1 0 U per ml and at pH 7-5. Endoglycosidase-F from Flavobacterium meningosepticum (EC 3.2.1.96) cleaves high mannose hybrid or complex saccharides from peptide chains. This enzyme was used at an activity of 1 U per ml and at pH 5 0. With the exception of neuraminidase, all other incubations were performed overnight at 37 'C. The glycosyl hydrolases were obtained from Boehringer Mannheim. Protease activity is not detectable in these. enzyme preparations by incubation of endoglycosidase F (6 U) or glycopeptidase F (20 U) with 200 ,ug of resorfin labelled casein overnight at 37 'C in appropriate buffer. The cleavage sites of the endoglycosidases on N-linked oligosaccharides are shown in Figure 2. Immunocytochemistry AGF4-48 and Leu-M 1 were used as neat hybridoma supernatant and the anti-blood group H Type II at a dilution of 1:10. Antibody binding to tissue sections was visualised by using both the avidin/biotin peroxidase complex method (Hsu, Raine & Fanger, 1981) and an indirect peroxidase method. The reagents used were obtained as ANA 170
154 S. FLEMING an ABC immunocytochemistry kit from Vector Laboratories and peroxidase-labelled rabbit and anti-mouse immunoglobulin (DAKO). Control experiments Buffer controls were performed by incubating sections with the buffer solutions which were used to perform the digestions of the glycosyl hydrolases. The specificity and sensitivity of the enzyme and immunocytochemical reactions were confirmed by performing simultaneous experiments on post-implantation mouse fetal tissue, a tissue in which the structure of the poly-N-acetyl lactosamine derivatives has been previously demonstrated using these techniques (Pennington, Rastan, Roelcke & Feizi, 1986). Standard immunocytochemistry controls were performed. These involved substituting an irrevelant monoclonal antibody (MAC 387) for the anti-glycosyl antibodies in the immunocytochemistry protocol. Additional controls involved addition of the ABC reagents only, without a primary antibody, to examine for endogenous biotin activity in the fixed tissue. In addition, the antigenic structure demonstrated by the binding of the monoclonal antibodies was confirmed by demonstrating the sensitivity of antibody binding to pre-digestion by the exoglycosidases, beta-galactosidase and alpha-fucosidase. These enzymes cleave from the carbohydrate chain the terminal galactoside and fucoside residues which confer the specificity of the LeX and H-Type II epitopes. In the immunocytochemical experiments, binding of the monoclonal antibodies was consistently sensitive to digestion of the tissue sections by these two enzymes. RESULTS
Examination of the haematoxylin and eosin-stained sections confirmed that the tissues examined were normal fetal kidneys with a capsule, subcapsular nephrogenic zone with immature nephrons and deeper cortex and medulla containing more mature nephrons. The different stages of nephrogenesis were observed in each fetal kidney. The distribution of LeX hapten and H- Type II antigen in human fetal kidney At the earliest stage of nephron development the LeX hapten was seen on the surface of the epithelial cells of the ureteric bud ampulla which was seen growing into the metanephric blastema (Fig. 3). No reactivity was seen in the metanephric blastema nor in the interstitial portions of the ureteric bud. LeX immunoreactivity persisted on the ampulla of the ureteric bud until after fusion of this structure with the distal tubular component of the S-shaped tubule (Fig. 4) but was then lost from this site. LeX reactivity was not seen in the renal vesicle nor on the S-shaped tubule. During maturation of the nephron, after fusion of the S-shaped tubule and ureteric bud, the activity for this antigen was seen on the proximal tubular epithelium. Staining was seen throughout the length of the proximal tubule initially but, at later stages, became confined to the epithelium of the pars recta (Fig. 5). At this stage of development no staining for LeX was observed in the collecting ducts, distal tubule or glomerulus. Reactivity was not exposed by pre-treatment with neuraminidase or alphagalactosidase. Reactivity of H-Type II substance was seen on the endothelial cells of the arteries, peritubular capillaries and glomerular capillaries. This staining was seen at all appropriate stages of development. In untreated sections this staining was weak, but pre-treatment of the sections with neuraminidase increased the strength of the staining
Oligosaccharides and renal organogenesis
...4
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(Fig. 6).Reactivity for H-Type II substance, after pre-treatment with neuraminidase,~~~~~~~~~~~~~~~~~~~~~~~~~~~~. wasalso seen in the mature collecting duct epithelium. H-Type. .I.substance was.not identifinieodtherureteric sitesoftheimanteuorprheronbud. Pre-treatmentwith~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~.... ~. alpha-galactosidasehadnthe oeffectdetection on of H-Type IIsubstance.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.......
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Oligosaccharides and renal organogenesis
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orangneshis ispe that whiuchtcurs betee the ingohyrotswhingurtricbu amspunilla and athgeundiffeyrentated mtantephi bumasTyem (Grobstein 1955, 1956).ce in this stdy,elpwe huave kdemnstaed thatee atnthisedsTagse moflephron develfuopmentthed LeXihaptien isN aexprsse lconamthe suFaeiof th epithelia the uretrnofexresicnbud amplosla.rlthis cels8lnin ben moexpressio persistsdeontrte during thesealstgsonphn developmentuniofother fusion ofth uareticulbud adthes S-eclshapedm tubue.epesdo h elsraeo el hc The 1-3lve alphatfucosyularitedrNacetyoslurn latsmirynepitoe whihopcontRstitut ethis.
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158
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of tissues (Hakomori et al. 1981; Feizi & Childs, 1985). The structure of the molecules carrying this epitope has been analysed by demonstrating their sensitivity to digestion by endoglycosidases. The molecule carrying LeX reactivity on the ureteric bud epithelium was sensitive to digestion by endo-beta-galactosidase. This enzyme digests linear forms of the poly-N-acetyl lactosamine backbone chain of these antigens (Scudder et al. 1984; Rastan et al. 1985). Using similar techniques, the molecules carrying LeX reactivity in mouse fetal tissues have been shown to be carried on a linear backbone of N-acetyl lactosamine (Rastan et al. 1985; Pennington et al. 1986). The immunoreactivity on the ureteric bud was also sensitive to digestion by glycopeptidase-F and endoglycosidase-F. Glycopeptidase-F hydrolyses the chemical bond between the first carbohydrate of an N-linked side chain and the asparagine residue in the protein (Plummer et al. 1984). Endoglycosidase-F digests carbohydrates of a non-bisected biantennary configuration (Elder & Alexander, 1982). These results suggest that the LeX hapten on the ureteric bud epithelium is carried on a molecule which has a linear poly-N-acetyl lactosamine sequence on a biantennary, non-bisected complex carbohydrate with is N-linked to a protein. Ozawa et al. (1985) have shown that the corresponding molecule on teratocarcinoma cells is a high molecular glycoprotein with N-linked saccharide structures. Precise identification of the glycoprotein carrying this antigenic determinant on the ureteric bud epithelium will require isolation and purification of the molecule. By contrast to the findings on the ureteric bud epithelium, LeX immunoreactivity on the proximal tubular epithelium was resistant to digestion by endoglycosidases. It has previously been suggested that the LeX hapten at this site is carried on a unique fucoganglioside (Rauvula, 1970). The results of the immunochemical experiments show that reactivity for the same monoclonal antibody can be carried on two different molecules on two anatomically distinct sites during human renal development. This has important implications for the study of development by immunological methods. The major site of H-Type II substance immunoreactivity in the developing kidney was on the vascular endothelium. The structure of the epitope identified by the H-Type II antibody is similar, if not identical, to the oligosaccharide recognised by the lectin from Ulex europaeus (UEA) (Robb & Stoddart, 1987). UEA reactivity has previously been demonstrated on the vascular endothelium of the developing kidney at sites similar to those in which we have found reactivity to the H-Type II substance (Holthofer, 1987). The immunoreactivity on the vascular endothelium was also sensitive to digestion by the endoglycosidases. This suggests that this molecule is also carried on a non-bisected biantennary N-linked saccharide. Robb & Stoddart (1987) have previously shown that the saccharide recognised by UEA is carried on a molecule of this type. H-Type II immunoreactivity on the collecting duct epithelium was found to be resistant to digestion by endoglycosidases. There was partial masking of some of these antigens by terminal sialylation. As has been previously shown, antigenic sites rendered cryptic in this way can be re-exposed by pre-treatment of tissue sections by neuraminidase (Howie & Brown, 1985). The precise significance of these developmentally regulated changes in the expression of this small series of defined carbohydrates remains, at the moment, obscure. These molecules have been shown to be constituent parts of differentiation antigens, receptor systems, and cell surface enzymes and their co-factors (Feizi, 1984; Feizi & Childs, 1985). An endogenous mammalian lectin which binds to the 1-3 alpha-fucosyl Nacetyl lactosamine which constitutes the LeX hapten has been described on
Oligosaccharides and renal organogenesis
159
hepatocytes (Prieels et al. 1975). Furthermore, Rastan et al. (1985) have shown that these related oligosaccharides are responsible for cell contact and compaction during the morula stage of the development of the pre-implantation murine embryo. Lectin-sugar interactions at the cell surface, mediated through changes in the expression of these carbohydrates, may function as signals during the induction of renal tubular differentiation. Alternatively, at the sites at which these oligosaccharides are carried on glycolipids they may be functioning as part of an enzyme co-factor system for transepithelial transport enzymes as has been shown previously for the Band 3 glycoprotein of erythrocytes (Feizi & Childs, 1985). In summary, by combining the specificity of monoclonal antibodies for the terminal portion of oligosaccharides with the specificity of glycosyl hydrolases for the internal chain structure, we have been able to perform an analysis of the structure of cell surface carbohydrates during the development of the human fetal kidney. These experiments have shown that there is a developmental site- and stage-specific pattern of changes in the expression of these molecules. SUMMARY
The structure of fucosylated derivatives of N-acetyl lactosamine expressed during human renal development has been studied by the combined use of monoclonal antibodies and glycosyl hydrolases. LeX is expressed on the ureteric bud ampulla carried N-linked on a glycoprotein. It is also present in the proximal tubule as a ganglioside. H-Type II substance is expressed on the endothelium of the developing renal vasculature. It is also carried N-linked to a glycoprotein. These regulated changes in the cell surface carbohydrate expression are important in understanding cell interactions and receptor systems during renal development. I wish to thank Mrs June Giddings for excellent technical support and Mrs Helena Black for secretarial assistance. REFERENCES ELDER, J. H. & ALEXANDER, S. (1982). Endo-beta-N-acetyl glucosaminidase-F: Endoglycosidase from Flavobacterium meningosepticum that cleaves high mannose and complex glycoproteins. Proceedings of the National Academy of Sciences of the USA 79, 4540-4544. FEIZI, T. (1984). Monoclonal antibodies reveal saccharide structures of glycoproteins as differentiation and tumour-associated antigens. Biochemical Society Transactions 12, 545-549. FEIZI, T. & CHILDS, R. S. (1985). Carbohydrate structures of glycoproteins and glycolipids as differentiation antigens, tumour-associated antigens and components of receptor systems. Trends in Biochemical Science 10, 24-29. FLEMING, S. & BROWN, G. (1986). Distribution of fucosylated N-acetyl lactosamine carbohydrate determinants during embryogenesis of the kidney in man. Histochemical Journal 18, 61-66. Fox, N., DAMJANOV, I., KNOWLES, B. B. & SOLTER, D. (1981). Immunohistochemical localisation of the early embryonic antigen (SSEA- 1) in post implantation mouse embryos and fetal and adult tissue. Developmental Biology 83, 391-398. Gooi, H. C., FEIZI, T., KAPADIA, A., KNOWLES, B. B., SOLTER, D. & EVANS, M. (1981). Stage specific embryonic antigen involves (1-3) fucosylated Type II blood group chains. Nature 292, 156-158. GROBSTEIN, C. (1955). Inductive interactions in the developing mouse metanephros. Journal of Experimental Zoology 130, 319-340. GROBSTEIN, C. (1956). Transfilter induction of tubules in mouse metanephric mesenchyme. Experimental Cell Research 10, 242-440. HAKOMORI, S., NUDELMAN, E., LEVEREY, S., SOLTER, D. & KNOWLES, B. B. (1981). The hapten structure of a developmentally regulated glycolipid antigen (SSEA-1) isolated from human erythrocytes and adenocarcinomas. Biochemical and Biophysical Research Communications 100, 1578-1586. HOLTHOFER, H. (1987). Vascularization of the embryonic kidney. Detection of endothelial cells with Ulex europeus I lectin. Cell Differentiation 20, 27-31.
160
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HOWIE, A. J. & BROWN, G. (1985). Effect of neuraminidase on the expression of the 3-fucosyl-N-acetyl lactosamine antigen in human tissues. Journal of Clinical Pathology 38, 409-418. Hsu, S. M., RAINE, L. & FANGER, H. (1981). Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques. Journal of Histochemistry and Cytochemistry 29, 577-580. JACOB, F. (1979). Cell surface and early stages of mouse embryogenesis. Current Topics in Developmental Biology 13, 117-135. KELLY, S. E. & FLEMING, S. (1987). The structure of fucosylated blood group substances in fetal rat skin. The combined use of monoclonal antibodies and glycosyl hydrolases. British Journal of Dermatology 118, 765-773. LLOYD, K. 0. (1987). Blood group antigens as markers for normal differentiation and malignant change. American Journal of Pathology 87, 129-139. OZAWA, M., MURAMATSU, T. & SOLTER, D. (1985). SSEA-1, a stage specific embryonic antigen of the mouse carried by the glycoprotein-bound large carbohydrate in embryonal carcinoma cells. Cell Differentiation 16, 160-173. PENNINGTON, S. E., RASTAN, S., ROELCKE, D. & FEIZI, T. (1986). Saccharide structures of the mouse embryo during the first 8 days of development. Inferences from immunocytochemical studies using monoclonal antibodies in conjunction with glycosidases. Journal of Embryology and Experimental Morphology 90, 335-361. PLUMMER, T. H., ELDER, J. H., ALEXANDER, S., PHELAN, A. W. & TAVENTINO, A. L. (1984). Demonstration of peptide N-glycosidase F activity in endo-beta-glucosaminidase F preparations. Journal of Biological Chemistry 259, 10700-10704. PRIEELS, J. P., PIZZOR, S. V., GLASGOW, L. R., PAULSON, J. C. & HILL, R. L. (1975). Hepatic receptor that specifically binds oligosaccharides containing fucosyl 1-3 N-acetyl glucosamine linkages. Proceedings of the National Academy of Sciences of the USA 75, 2215-2219. RASTAN, S., THORPE, S. J., SCUDDER, P., BROWN, S., GooI, H. C. & FEIZI, T. (1985). Cell interactions in preimplantation embryos. Evidence for involvement of saccharides of the poly-N-acetyl lactosamine series. Journal of Embryology and Experimental Morphology 87, 115-128. RAUVULA, M. (1970). The fucoganglioside of the human kidney. FEBS Letters 62, 161-164. ROBB, J. L. & STODDART, R. W. (1987). N-linked oligosaccharide sequences of endothelial cells. Journal of Pathology 152, 242A. SCUDDER, P., HORFLAND, P., UEMURA, K. & FEIZI, T. (1984). Endogalactosidase of Bacterioides fragilis and Escherichia freunoii hydrolyse linear but not branched oligosaccharide domains of glycolipids of the neolacto series. Journal of Biological Chemistry 259, 6596-6592. SOLTER, D. & KNOWLES, B. B. (1979). Developmental stage specific antigens during mouse embryogenesis. Current Topics in Developmental Biology 13, 139-165.
J. Anat. (1990), 170, pp. 151-160 With 6 figures Printed in Great Britain
N-linked oligosaccharides during human renal organogenesis S. FLEMING*
University Department of Pathology, Southampton General Hospital, Southampton S09 4XY
(Accepted 18 December 1989) INTRODUCTION
There is interest in the developmental stage specific expression of cell surface carbohydrates which may have a function in cell interactions during embryogenesis. The cell surface expression of fucosylated derivatives of poly-N-acetyl lactosamine has particularly been shown to be important during mammalian development (Feizi & Childs, 1985). These glycoconjugates have been shown to be expressed in a characteristic distribution (Solter & Knowles, 1979), and to be involved in cell interactions at the morula stage and during gastrulation of pre-implantation mammalian embryos (Jacob, 1979). Related molecules are expressed on the cell surface of some cells during the differentiation of fetal tissues (Kelly & Fleming, 1987). Fleming & Brown (1986) have recently shown that fucosylated derivatives of N-acetyl lactosamine are expressed at important sites of organogenetic interactions during the development of the human kidney. Demonstration of these cell surface molecules has been achieved by utilising the specificity of monoclonal antibodies (Lloyd, 1987). The monoclonal antibodies used in these investigations are specific for the terminal oligosaccharide epitopes which are carried on larger carbohydrate side chains of glycolipids or glycoproteins (Feizi, 1984). Although these molecules can be carried on either lipids or proteins, Ozawa, Muramatsu & Solter (1985) have shown that during important stages of embryogenesis the saccharides are frequently carried on a large molecular weight glycoprotein which they have called embryoglycan. It is now possible to analyse parts of the structure of the remainder of the carbohydrate side chains and their linkages to peptides by the use of specific glycosyl hydrolases (Rastan et al. 1985). These are enzymes, mostly of plant or bacterial origin, which hydrolyse linkages of a specific configuration between defined saccharides. Thus, by testing the sensitivity of monoclonal antibody binding in immunocytochemical experiments to pre-digestion by specific glycosyl hydrolases, the structure of the carbohydrate side chain carrying the appropriate epitopes may be inferred. We have studied the distribution of fucosylated derivatives of the Type II blood group substance during the development and differentiation of the human kidney using monoclonal antibodies and the immunoperoxidase technique. We have further analysed the structure of these glycoconjugates by using glycosyl hydrolases.
* Present address: Department of Pathology, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, Scotland.
152
S. FLEMING
LeX
P-Gal 1-4 GLCNAC 3
FUC
H-type 11
«-Gal 1-4 GLCNAC 2
FUC Fig. 1. The structure of the oligosaccharides conferring LeX and H-Type II antigenicity. MATERIALS AND METHODS
Tissues Both cryostat-sectioned and formalin-fixed, paraffin-embedded tissues were used. Similar results were obtained with both types of tissue. Fetal kidneys were dissected from fetuses of gestational ages between 12 and 18 weeks which were obtained during legal terminations of pregnancy. For cryostat sectioning a bisected fetal kidney was snap-frozen in liquid nitrogen and stored at -70°C until use. Six micron thick cryostat sections were cut, one section of which was stained with haematoxylin and eosin. Multiple adjacent cells were used for immunocytochemical experiments after brief fixation in cold acetone. From each fetus the second kidney was fixed in 10% neutral buffered formalin for 24 hours. The tissues were then processed to paraffin wax and 4 ,um sections cut. They were stained with haematoxylin and eosin and multiple adjacent sections were used in immunocytochemical experiments. Ten specimens of each type of tissue were used in these experiments. Antibodies The murine monoclonal antibodies, AGF4-48 and Leu-M 1, are both reactive against the LeX hapten. The structure of this antigenic determinant is shown in Figure 1. This oligosaccharide is also responsible for the reactivity of the antibody, SSEA-1, and the human leucocyte marker, CD15 (Gooi et al. 1981). Both AGF4-48 and LeuMl show CD 15-like immunoreactivity in their tissue distribution and their specificity for the above oligosaccharide has been confirmed by competitive inhibition of binding, using synthetic oligosaccharides (Kelly & Fleming, 1987). A commercially available (DAKO) antibody to human blood group Substance H on a Type II precursor chain was also used. The epitope recognised by this antibody is illustrated in Figure 1. All three of these antibodies are murine IgM molecules.
Glycosyl hydrolases Several glycosyl hydrolases were used. Sections were incubated with these enzymes before incubation with the primary antibodies in the immunocytochemical technique. Neuraminidase (EC 3.2. 1 .18) from Clostridium perfringens removed terminal neuraminic acid from carbohydrate chains. Tissue sections were incubated with neuraminidase for 2 hours at room temperature and at pH 4-6. The structure of the poly-N-acetyl lactosamine carbohydrate backbone of the
Oligosaccharides and renal organogenesis [21
Fucosidase
NEU
FUC ± GAL
GAL
.
GLCNAC I GAL
GLCNAC 4GAL
-
153
Neuroaminidase
Endogalactosidase
l
GLCNAC
GLCNAC
l MAN
MAN
MAN
GLCNAC
4-
Endoglycosidase F
GLCNAC %
Glycopeptidase F
ASN Fig. 2. The cleavage sites on N-linked oligosaccharides of the endoglycosidases.
antigens can be inferred from the sensitivity to digestion by endo-beta-galactosidase (Scudder, Horfland, Ibemura & Feizi, 1984). This enzyme will digest linear chains of galactose and N-acetyl-glucosamine but will not digest branched chains of the same carbohydrates. It, therefore, distinguishes between the human blood group antigen, i, which is enzyme-sensitive, and blood group, I, which is enzyme-resistant. Endo-betagalactosidase (EC 3.2.1.103) purified from Bacterioides fragilis by the method of Scudder et al. (1984) was incubated with tissue sections at an activity of 0 1 U per ml and at pH 5-8. N-linked oligosaccharides are sensitive to digestion by glycopeptidase-F. This enzyme cleaves high mannose hybrid and complex glycans from glycoproteins between the ASN of the peptide and the GLCNAC of the carbohydrate (Plummer et al. 1984). The glycopeptidase-F (EC 3.2.2.18) used in these experiments was purified from Flavobacterium meningosepticum and was used at an activity of 1 0 U per ml and at pH 7-5. Endoglycosidase-F from Flavobacterium meningosepticum (EC 3.2.1.96) cleaves high mannose hybrid or complex saccharides from peptide chains. This enzyme was used at an activity of 1 U per ml and at pH 5 0. With the exception of neuraminidase, all other incubations were performed overnight at 37 'C. The glycosyl hydrolases were obtained from Boehringer Mannheim. Protease activity is not detectable in these. enzyme preparations by incubation of endoglycosidase F (6 U) or glycopeptidase F (20 U) with 200 ,ug of resorfin labelled casein overnight at 37 'C in appropriate buffer. The cleavage sites of the endoglycosidases on N-linked oligosaccharides are shown in Figure 2. Immunocytochemistry AGF4-48 and Leu-M 1 were used as neat hybridoma supernatant and the anti-blood group H Type II at a dilution of 1:10. Antibody binding to tissue sections was visualised by using both the avidin/biotin peroxidase complex method (Hsu, Raine & Fanger, 1981) and an indirect peroxidase method. The reagents used were obtained as ANA 170
154 S. FLEMING an ABC immunocytochemistry kit from Vector Laboratories and peroxidase-labelled rabbit and anti-mouse immunoglobulin (DAKO). Control experiments Buffer controls were performed by incubating sections with the buffer solutions which were used to perform the digestions of the glycosyl hydrolases. The specificity and sensitivity of the enzyme and immunocytochemical reactions were confirmed by performing simultaneous experiments on post-implantation mouse fetal tissue, a tissue in which the structure of the poly-N-acetyl lactosamine derivatives has been previously demonstrated using these techniques (Pennington, Rastan, Roelcke & Feizi, 1986). Standard immunocytochemistry controls were performed. These involved substituting an irrevelant monoclonal antibody (MAC 387) for the anti-glycosyl antibodies in the immunocytochemistry protocol. Additional controls involved addition of the ABC reagents only, without a primary antibody, to examine for endogenous biotin activity in the fixed tissue. In addition, the antigenic structure demonstrated by the binding of the monoclonal antibodies was confirmed by demonstrating the sensitivity of antibody binding to pre-digestion by the exoglycosidases, beta-galactosidase and alpha-fucosidase. These enzymes cleave from the carbohydrate chain the terminal galactoside and fucoside residues which confer the specificity of the LeX and H-Type II epitopes. In the immunocytochemical experiments, binding of the monoclonal antibodies was consistently sensitive to digestion of the tissue sections by these two enzymes. RESULTS
Examination of the haematoxylin and eosin-stained sections confirmed that the tissues examined were normal fetal kidneys with a capsule, subcapsular nephrogenic zone with immature nephrons and deeper cortex and medulla containing more mature nephrons. The different stages of nephrogenesis were observed in each fetal kidney. The distribution of LeX hapten and H- Type II antigen in human fetal kidney At the earliest stage of nephron development the LeX hapten was seen on the surface of the epithelial cells of the ureteric bud ampulla which was seen growing into the metanephric blastema (Fig. 3). No reactivity was seen in the metanephric blastema nor in the interstitial portions of the ureteric bud. LeX immunoreactivity persisted on the ampulla of the ureteric bud until after fusion of this structure with the distal tubular component of the S-shaped tubule (Fig. 4) but was then lost from this site. LeX reactivity was not seen in the renal vesicle nor on the S-shaped tubule. During maturation of the nephron, after fusion of the S-shaped tubule and ureteric bud, the activity for this antigen was seen on the proximal tubular epithelium. Staining was seen throughout the length of the proximal tubule initially but, at later stages, became confined to the epithelium of the pars recta (Fig. 5). At this stage of development no staining for LeX was observed in the collecting ducts, distal tubule or glomerulus. Reactivity was not exposed by pre-treatment with neuraminidase or alphagalactosidase. Reactivity of H-Type II substance was seen on the endothelial cells of the arteries, peritubular capillaries and glomerular capillaries. This staining was seen at all appropriate stages of development. In untreated sections this staining was weak, but pre-treatment of the sections with neuraminidase increased the strength of the staining
Oligosaccharides and renal organogenesis
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orangneshis ispe that whiuchtcurs betee the ingohyrotswhingurtricbu amspunilla and athgeundiffeyrentated mtantephi bumasTyem (Grobstein 1955, 1956).ce in this stdy,elpwe huave kdemnstaed thatee atnthisedsTagse moflephron develfuopmentthed LeXihaptien isN aexprsse lconamthe suFaeiof th epithelia the uretrnofexresicnbud amplosla.rlthis cels8lnin ben moexpressio persistsdeontrte during thesealstgsonphn developmentuniofother fusion ofth uareticulbud adthes S-eclshapedm tubue.epesdo h elsraeo el hc The 1-3lve alphatfucosyularitedrNacetyoslurn latsmirynepitoe whihopcontRstitut ethis.
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158
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of tissues (Hakomori et al. 1981; Feizi & Childs, 1985). The structure of the molecules carrying this epitope has been analysed by demonstrating their sensitivity to digestion by endoglycosidases. The molecule carrying LeX reactivity on the ureteric bud epithelium was sensitive to digestion by endo-beta-galactosidase. This enzyme digests linear forms of the poly-N-acetyl lactosamine backbone chain of these antigens (Scudder et al. 1984; Rastan et al. 1985). Using similar techniques, the molecules carrying LeX reactivity in mouse fetal tissues have been shown to be carried on a linear backbone of N-acetyl lactosamine (Rastan et al. 1985; Pennington et al. 1986). The immunoreactivity on the ureteric bud was also sensitive to digestion by glycopeptidase-F and endoglycosidase-F. Glycopeptidase-F hydrolyses the chemical bond between the first carbohydrate of an N-linked side chain and the asparagine residue in the protein (Plummer et al. 1984). Endoglycosidase-F digests carbohydrates of a non-bisected biantennary configuration (Elder & Alexander, 1982). These results suggest that the LeX hapten on the ureteric bud epithelium is carried on a molecule which has a linear poly-N-acetyl lactosamine sequence on a biantennary, non-bisected complex carbohydrate with is N-linked to a protein. Ozawa et al. (1985) have shown that the corresponding molecule on teratocarcinoma cells is a high molecular glycoprotein with N-linked saccharide structures. Precise identification of the glycoprotein carrying this antigenic determinant on the ureteric bud epithelium will require isolation and purification of the molecule. By contrast to the findings on the ureteric bud epithelium, LeX immunoreactivity on the proximal tubular epithelium was resistant to digestion by endoglycosidases. It has previously been suggested that the LeX hapten at this site is carried on a unique fucoganglioside (Rauvula, 1970). The results of the immunochemical experiments show that reactivity for the same monoclonal antibody can be carried on two different molecules on two anatomically distinct sites during human renal development. This has important implications for the study of development by immunological methods. The major site of H-Type II substance immunoreactivity in the developing kidney was on the vascular endothelium. The structure of the epitope identified by the H-Type II antibody is similar, if not identical, to the oligosaccharide recognised by the lectin from Ulex europaeus (UEA) (Robb & Stoddart, 1987). UEA reactivity has previously been demonstrated on the vascular endothelium of the developing kidney at sites similar to those in which we have found reactivity to the H-Type II substance (Holthofer, 1987). The immunoreactivity on the vascular endothelium was also sensitive to digestion by the endoglycosidases. This suggests that this molecule is also carried on a non-bisected biantennary N-linked saccharide. Robb & Stoddart (1987) have previously shown that the saccharide recognised by UEA is carried on a molecule of this type. H-Type II immunoreactivity on the collecting duct epithelium was found to be resistant to digestion by endoglycosidases. There was partial masking of some of these antigens by terminal sialylation. As has been previously shown, antigenic sites rendered cryptic in this way can be re-exposed by pre-treatment of tissue sections by neuraminidase (Howie & Brown, 1985). The precise significance of these developmentally regulated changes in the expression of this small series of defined carbohydrates remains, at the moment, obscure. These molecules have been shown to be constituent parts of differentiation antigens, receptor systems, and cell surface enzymes and their co-factors (Feizi, 1984; Feizi & Childs, 1985). An endogenous mammalian lectin which binds to the 1-3 alpha-fucosyl Nacetyl lactosamine which constitutes the LeX hapten has been described on
Oligosaccharides and renal organogenesis
159
hepatocytes (Prieels et al. 1975). Furthermore, Rastan et al. (1985) have shown that these related oligosaccharides are responsible for cell contact and compaction during the morula stage of the development of the pre-implantation murine embryo. Lectin-sugar interactions at the cell surface, mediated through changes in the expression of these carbohydrates, may function as signals during the induction of renal tubular differentiation. Alternatively, at the sites at which these oligosaccharides are carried on glycolipids they may be functioning as part of an enzyme co-factor system for transepithelial transport enzymes as has been shown previously for the Band 3 glycoprotein of erythrocytes (Feizi & Childs, 1985). In summary, by combining the specificity of monoclonal antibodies for the terminal portion of oligosaccharides with the specificity of glycosyl hydrolases for the internal chain structure, we have been able to perform an analysis of the structure of cell surface carbohydrates during the development of the human fetal kidney. These experiments have shown that there is a developmental site- and stage-specific pattern of changes in the expression of these molecules. SUMMARY
The structure of fucosylated derivatives of N-acetyl lactosamine expressed during human renal development has been studied by the combined use of monoclonal antibodies and glycosyl hydrolases. LeX is expressed on the ureteric bud ampulla carried N-linked on a glycoprotein. It is also present in the proximal tubule as a ganglioside. H-Type II substance is expressed on the endothelium of the developing renal vasculature. It is also carried N-linked to a glycoprotein. These regulated changes in the cell surface carbohydrate expression are important in understanding cell interactions and receptor systems during renal development. I wish to thank Mrs June Giddings for excellent technical support and Mrs Helena Black for secretarial assistance. REFERENCES ELDER, J. H. & ALEXANDER, S. (1982). Endo-beta-N-acetyl glucosaminidase-F: Endoglycosidase from Flavobacterium meningosepticum that cleaves high mannose and complex glycoproteins. Proceedings of the National Academy of Sciences of the USA 79, 4540-4544. FEIZI, T. (1984). Monoclonal antibodies reveal saccharide structures of glycoproteins as differentiation and tumour-associated antigens. Biochemical Society Transactions 12, 545-549. FEIZI, T. & CHILDS, R. S. (1985). Carbohydrate structures of glycoproteins and glycolipids as differentiation antigens, tumour-associated antigens and components of receptor systems. Trends in Biochemical Science 10, 24-29. FLEMING, S. & BROWN, G. (1986). Distribution of fucosylated N-acetyl lactosamine carbohydrate determinants during embryogenesis of the kidney in man. Histochemical Journal 18, 61-66. Fox, N., DAMJANOV, I., KNOWLES, B. B. & SOLTER, D. (1981). Immunohistochemical localisation of the early embryonic antigen (SSEA- 1) in post implantation mouse embryos and fetal and adult tissue. Developmental Biology 83, 391-398. Gooi, H. C., FEIZI, T., KAPADIA, A., KNOWLES, B. B., SOLTER, D. & EVANS, M. (1981). Stage specific embryonic antigen involves (1-3) fucosylated Type II blood group chains. Nature 292, 156-158. GROBSTEIN, C. (1955). Inductive interactions in the developing mouse metanephros. Journal of Experimental Zoology 130, 319-340. GROBSTEIN, C. (1956). Transfilter induction of tubules in mouse metanephric mesenchyme. Experimental Cell Research 10, 242-440. HAKOMORI, S., NUDELMAN, E., LEVEREY, S., SOLTER, D. & KNOWLES, B. B. (1981). The hapten structure of a developmentally regulated glycolipid antigen (SSEA-1) isolated from human erythrocytes and adenocarcinomas. Biochemical and Biophysical Research Communications 100, 1578-1586. HOLTHOFER, H. (1987). Vascularization of the embryonic kidney. Detection of endothelial cells with Ulex europeus I lectin. Cell Differentiation 20, 27-31.
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HOWIE, A. J. & BROWN, G. (1985). Effect of neuraminidase on the expression of the 3-fucosyl-N-acetyl lactosamine antigen in human tissues. Journal of Clinical Pathology 38, 409-418. Hsu, S. M., RAINE, L. & FANGER, H. (1981). Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques. Journal of Histochemistry and Cytochemistry 29, 577-580. JACOB, F. (1979). Cell surface and early stages of mouse embryogenesis. Current Topics in Developmental Biology 13, 117-135. KELLY, S. E. & FLEMING, S. (1987). The structure of fucosylated blood group substances in fetal rat skin. The combined use of monoclonal antibodies and glycosyl hydrolases. British Journal of Dermatology 118, 765-773. LLOYD, K. 0. (1987). Blood group antigens as markers for normal differentiation and malignant change. American Journal of Pathology 87, 129-139. OZAWA, M., MURAMATSU, T. & SOLTER, D. (1985). SSEA-1, a stage specific embryonic antigen of the mouse carried by the glycoprotein-bound large carbohydrate in embryonal carcinoma cells. Cell Differentiation 16, 160-173. PENNINGTON, S. E., RASTAN, S., ROELCKE, D. & FEIZI, T. (1986). Saccharide structures of the mouse embryo during the first 8 days of development. Inferences from immunocytochemical studies using monoclonal antibodies in conjunction with glycosidases. Journal of Embryology and Experimental Morphology 90, 335-361. PLUMMER, T. H., ELDER, J. H., ALEXANDER, S., PHELAN, A. W. & TAVENTINO, A. L. (1984). Demonstration of peptide N-glycosidase F activity in endo-beta-glucosaminidase F preparations. Journal of Biological Chemistry 259, 10700-10704. PRIEELS, J. P., PIZZOR, S. V., GLASGOW, L. R., PAULSON, J. C. & HILL, R. L. (1975). Hepatic receptor that specifically binds oligosaccharides containing fucosyl 1-3 N-acetyl glucosamine linkages. Proceedings of the National Academy of Sciences of the USA 75, 2215-2219. RASTAN, S., THORPE, S. J., SCUDDER, P., BROWN, S., GooI, H. C. & FEIZI, T. (1985). Cell interactions in preimplantation embryos. Evidence for involvement of saccharides of the poly-N-acetyl lactosamine series. Journal of Embryology and Experimental Morphology 87, 115-128. RAUVULA, M. (1970). The fucoganglioside of the human kidney. FEBS Letters 62, 161-164. ROBB, J. L. & STODDART, R. W. (1987). N-linked oligosaccharide sequences of endothelial cells. Journal of Pathology 152, 242A. SCUDDER, P., HORFLAND, P., UEMURA, K. & FEIZI, T. (1984). Endogalactosidase of Bacterioides fragilis and Escherichia freunoii hydrolyse linear but not branched oligosaccharide domains of glycolipids of the neolacto series. Journal of Biological Chemistry 259, 6596-6592. SOLTER, D. & KNOWLES, B. B. (1979). Developmental stage specific antigens during mouse embryogenesis. Current Topics in Developmental Biology 13, 139-165.
