Molecular Cloning and Characterization of Chick Sialoprotein Associated with Cones and Rods, a Developmentally Regulated Glycoprotein of Interphotoreceptor Matrix
2002; Elsevier BV; Volume: 277; Issue: 28 Linguagem: Inglês
10.1074/jbc.m201279200
ISSN1083-351X
AutoresMasahiro Zako, Masayoshi Iwaki, Masahiko Yoneda, Osamu Miyaishi, Jinsong Zhao, Yasuhiko Suzuki, Makoto Takeuchi, Goichiro Miyake, Hiroshi Ikagawa, Koji Kimata,
Tópico(s)Cell Adhesion Molecules Research
ResumoMY-174 is an IgM class monoclonal antibody originally established against chick PG-M/versican. The antibody specifically stains the photoreceptor layer, where we recently reported an absence of PG-M/versican. In this study, we re-characterized the antibody and identified the molecule that reacts to MY-174 at the photoreceptor layer. Immunohistochemistry localized the antigen to the matrix surrounding photoreceptors. A variety of glycosidase digestions showed that the antigen is the 150-kDa glycoprotein that has sialylatedN- and O-linked glycoconjugates having a molecular mass of more than 30-kDa. The peptide sequences obtained from purified MY-174 antigen showed we had sequenced a full-length cDNA with an open reading frame of 2787 base pairs, encoding a polypeptide of 928 amino acids, with 56 and 54% identities to human and mouse sialoprotein associated with cones and rods (SPACRs), respectively, and with the structural features observed in SPACRs. The specific sialylated O-glycoconjugates here are involved in the epitope structure for MY-174. SPACR first appeared by embryonic days 15–16, and expression increased with developmental age, paralleling the adhesion between neural retina and retinal pigment epithelium. Thus, we concluded that the MY-174 antigen at the photoreceptor layer, a developmentally regulated glycoprotein, is identical to chick SPACR and may be involved in a novel system mediating adhesion between neural retina and retinal pigment epithelium. MY-174 is an IgM class monoclonal antibody originally established against chick PG-M/versican. The antibody specifically stains the photoreceptor layer, where we recently reported an absence of PG-M/versican. In this study, we re-characterized the antibody and identified the molecule that reacts to MY-174 at the photoreceptor layer. Immunohistochemistry localized the antigen to the matrix surrounding photoreceptors. A variety of glycosidase digestions showed that the antigen is the 150-kDa glycoprotein that has sialylatedN- and O-linked glycoconjugates having a molecular mass of more than 30-kDa. The peptide sequences obtained from purified MY-174 antigen showed we had sequenced a full-length cDNA with an open reading frame of 2787 base pairs, encoding a polypeptide of 928 amino acids, with 56 and 54% identities to human and mouse sialoprotein associated with cones and rods (SPACRs), respectively, and with the structural features observed in SPACRs. The specific sialylated O-glycoconjugates here are involved in the epitope structure for MY-174. SPACR first appeared by embryonic days 15–16, and expression increased with developmental age, paralleling the adhesion between neural retina and retinal pigment epithelium. Thus, we concluded that the MY-174 antigen at the photoreceptor layer, a developmentally regulated glycoprotein, is identical to chick SPACR and may be involved in a novel system mediating adhesion between neural retina and retinal pigment epithelium. interphotoreceptor matrix sialoprotein associated with cones and rods phosphate-buffered saline epidermal growth factor embryonic day newborn adult receptor for hyaluronic acid-mediated motility MY-174, a monoclonal antibody, has been shown to recognize chick PG-M/versican (1Yamagata M. Shinomura T. Kimata K. Anat. Embryol. 1993; 187: 433-444Crossref PubMed Scopus (82) Google Scholar), a member of the hyaluronan-binding chondroitin sulfate proteoglycan family (2Kimata K. Oike Y. Tani K. Shinomura T. Yamagata M. Uritani M. Suzuki S. J. Biol. Chem. 1986; 261: 13517-13525Abstract Full Text PDF PubMed Google Scholar, 3Zimmermann D.R. Ruoslahti E. EMBO J. 1989; 8: 2975-2981Crossref PubMed Scopus (499) Google Scholar, 4Shinomura T. Nishida Y. Ito K. Kimata K. J. Biol. Chem. 1993; 268: 14461-14469Abstract Full Text PDF PubMed Google Scholar). In the eye, MY-174 specifically stains the photoreceptor layer (1Yamagata M. Shinomura T. Kimata K. Anat. Embryol. 1993; 187: 433-444Crossref PubMed Scopus (82) Google Scholar). However, recently we demonstrated an absence of PG-M/versican at the chick photoreceptor layer using a polyclonal antibody that recognizes all alternatively spliced forms of PG-M/versican (5Zako M. Shinomura T. Miyaishi O. Iwaki M. Kimata K. J. Neurochem. 1997; 69: 2155-2161Crossref PubMed Scopus (32) Google Scholar). These inconsistent results suggest that MY-174 does not recognize PG-M/versican but another molecule in the photoreceptor layer.The IPM (interphotoreceptormatrix),1 resides in an extracellular compartment between the outer limiting membrane of the neural retina and apical surface of the retinal pigment epithelium and is composed of proteins, glycoproteins, proteoglycans, and glycosaminoglycans (6Adler A. Severin K.M. Exp. Eye Res. 1981; 32: 755-769Crossref PubMed Scopus (91) Google Scholar, 7Adler A. Klucznik K.M. Exp. Eye Res. 1982; 34: 423-434Crossref PubMed Scopus (98) Google Scholar). A variety of important reactions relating to vision, including visual pigment chromophore exchange, metabolite trafficking, photoreceptor alignment, and membrane turnover, is thought to be mediated by the IPM (8Hageman G.S. Johnson L.V. Osborne N.N. Chader G.J. Progress in Retinal Research. Pergamon, Oxford1991: 207-249Google Scholar). However, one of the most essential functions of IPM is that of a biological glue for retinal adhesion through its viscous adhesive properties (9Zauberman H. Berman E.R. Exp. Eye Res. 1969; 8: 276-283Crossref PubMed Google Scholar, 10Zauberman H. Zinn K.M. Marmor M.F. The Retinal Pigment Epithelium. Harvard University Press, Cambridge1979: 192-204Google Scholar). Retinal adhesiveness can be weakened by treatments with enzymes such as neuraminidase, hyaluronidase, and chondroitinase ABC (11Yao X.Y. Hageman G.S. Marmor M.F. Invest. Ophthalmol. Vis. Sci. 1990; 31: 2051-2058PubMed Google Scholar, 12Yao X.Y. Hageman G.S. Marmor M.F. Invest. Ophthalmol. Vis. Sci. 1992; 33: 498-503PubMed Google Scholar). Intracellular blocking of glycosylation with xyloside also prevents the secretion of proteoglycans and results in retinal detachment (13Lazarus H.S. Hageman G.S. Invest. Ophthalmol. Vis. Sci. 1992; 33: 364-376PubMed Google Scholar). These results suggest that the proteoglycans, glycosaminoglycans, or glycoproteins of the IPM are involved in retinal adhesion. However, the specific IPM molecule that mediates adhesion has not been identified.SPACR (sialoprotein associated withcones and rods) is a hyaluronan-binding glycoprotein newly identified in adult human PBS (phosphate-buffered saline)-insoluble IPM (14Hollyfield J.G. Rayborn M.E. Landers R.A. Myers K.M. Exp. Eye Res. 1990; 51: 107-110Crossref PubMed Scopus (43) Google Scholar, 15Tien L. Rayborn M.E. Hollyfield J.G. Exp. Eye Res. 1992; 55: 297-306Crossref PubMed Scopus (32) Google Scholar, 16Acharya S. Rayborn M.E. Hollyfield J.G. Glycobiology. 1998; 8: 997-1006Crossref PubMed Scopus (35) Google Scholar, 17Acharya S. Rodriguez I.R. Moreira E.F. Midura R.J. Misono K. Todres E. Hollyfield J.G. J. Biol. Chem. 1998; 273: 31599-31606Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). This 147- to 150-kDa glycoprotein was purified by wheat germ agglutinin-affinity chromatography and characterized (17Acharya S. Rodriguez I.R. Moreira E.F. Midura R.J. Misono K. Todres E. Hollyfield J.G. J. Biol. Chem. 1998; 273: 31599-31606Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). It has been shown that (a) SPACR is heavily sialylated, (b) both N- andO-linked glycoconjugates are present in the molecule, and (c) glycoconjugates account for ∼30% of the molecular mass (17Acharya S. Rodriguez I.R. Moreira E.F. Midura R.J. Misono K. Todres E. Hollyfield J.G. J. Biol. Chem. 1998; 273: 31599-31606Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Recently, mouse SPACR was also cloned and characterized (18Lee J.W. Chen Q. Rayborn M.E. Shadrach K.G. Crabb J.W. Rodriguez I.R. Hollyfield J.G. Exp. Eye Res. 2000; 71: 341-352Crossref PubMed Scopus (14) Google Scholar). Both human and mouse SPACRs have a large central mucin-like domain flanked by consensus sites for N-linked oligosaccharide attachment, one EGF-like domain near the C-terminal, and several potential hyaluronan-binding motifs in common. Interestingly, biochemical studies showed that SPACR is a glycoprotein in human and a proteoglycan in mouse. Except for the ability of SPACR to bind hyaluronan (17Acharya S. Rodriguez I.R. Moreira E.F. Midura R.J. Misono K. Todres E. Hollyfield J.G. J. Biol. Chem. 1998; 273: 31599-31606Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), other properties or functional roles of SPACR remain unknown.In the present study, we first examined the localizations of the retinal antigen that binds to MY-174 in the photoreceptor layer. We then determined a full-length cDNA of MY-174 antigen at photoreceptor layer and showed that the antigen corresponded to chick SPACR. Finally, to investigate the biological significance of SPACR, we compared the expressions of chick SPACR and the occurrence of adhesion between neural retina and retinal pigment epithelium during development.DISCUSSIONHere we showed that a MY-174 antigen in the IPM is the ortholog of human SPACR and that there is an increase in SPACR expression during chick development. We also showed that the expression increases in parallel with retinal adhesiveness, implying that SPACR might be involved in the adhesion between neural retina and retinal pigment epithelium.We showed that sialylated O-linked glycoconjugates are involved in the epitope structure recognized by MY-174, which was developed as a monoclonal antibody to the core protein of PG-M/versican. The antigen was prepared by mild alkali treatment of the core protein of PG-M/versican, and the treatment eliminated glycoconjugates from the core protein (1Yamagata M. Shinomura T. Kimata K. Anat. Embryol. 1993; 187: 433-444Crossref PubMed Scopus (82) Google Scholar). However, when we made fusion proteins for the full length of PG-M/versican (V0) by Escherichia coli, MY-174 showed no immunoreactivity to the fusion core protein (data not shown). No immunoreactive clones could be detected by a MY-174 in screening of the chick retina cDNA library (data not shown). Acharya et al. (16Acharya S. Rayborn M.E. Hollyfield J.G. Glycobiology. 1998; 8: 997-1006Crossref PubMed Scopus (35) Google Scholar) mentioned that the major glycoconjugate on SPACR is the O-linked carbohydrate, NeuAcα2–3Galβ1–3GalNAc and that the O-linked sugar in SPACR could have a structure similar to that present in bone sialoprotein and aggrecan. At this stage we speculate that any residualO-glycans left on the core protein of PG-M/versican, specific but similar to those characteristic of SPACR, were the antigen for MY-174.Because our previous study using a polyclonal antibody that recognizes all PG-M/versican forms showed no staining in the photoreceptor layers of adult chick retinas (5Zako M. Shinomura T. Miyaishi O. Iwaki M. Kimata K. J. Neurochem. 1997; 69: 2155-2161Crossref PubMed Scopus (32) Google Scholar), we also thought it unlikely that PG-M/versican would be a target for MY-174 in retina. We have repeated this published observation with 2B1, a monoclonal antibody that recognizes all human PG-M/versican forms (25Isogai Z. Shinomura T. Yamakawa N. Takeuchi J. Tsuji T. Heinegård D. Kimata K. Cancer Res. 1996; 56: 3902-3908PubMed Google Scholar), but again no specific staining of the human photoreceptor layer could be detected (data not shown). Furthermore, no specific staining at an adult mouse photoreceptor layer could be detected using polyclonal antibodies against mouse PG-M/versican (data not shown).MY-174 staining showed resistance to a combination of neuraminidase andO-glycanase treatments around the outer segments of photoreceptor cells. Tien et al. (15Tien L. Rayborn M.E. Hollyfield J.G. Exp. Eye Res. 1992; 55: 297-306Crossref PubMed Scopus (32) Google Scholar) also reported that wheat germ agglutinin staining of SPACR is resistant to neuraminidase around the outer segments of photoreceptor cells in retinal sections. The reason for the resistance to enzymatic treatments is unclear, but the IPM seems to have different properties around inner or outer segments of photoreceptor cells.Acharya et al. (17Acharya S. Rodriguez I.R. Moreira E.F. Midura R.J. Misono K. Todres E. Hollyfield J.G. J. Biol. Chem. 1998; 273: 31599-31606Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) showed human SPACR has a functional hyaluronan-binding domain. This domain (RHAMM-type hyaluronan binding motif) corresponded to 280KEIHVLGFK288 in chick SPACR. It showed high consensus with human and mouse SPACR. This is a candidate for the RHAMM-type hyaluronan binding motif, because a single acidic group can be present in one residue inside either flanking basic residue in a B7XB RHAMM-type hyaluronan binding motif (26Yang B. Yang B.L. Savani R.C. Turley E.A. EMBO J. 1994; 13: 286-296Crossref PubMed Scopus (334) Google Scholar). Our data showed chick SPACR can bind hyaluronan. Mutagenesis studies of the putative hyaluronan binding motif will be required to establish the site of hyaluronan binding in chick SPACR. Because hyaluronan is a prominent constituent of the IPM in all species studied except mouse (27Hollyfield J.G. Rayborn M.E. Tammi R. Exp. Eye Res. 1997; 65: 603-608Crossref PubMed Scopus (18) Google Scholar, 28Hollyfield J.G. Rayborn M.E. Tammi M. Tammi R. Exp. Eye Res. 1998; 66: 241-248Crossref PubMed Scopus (44) Google Scholar), we speculate that SPACR and hyaluronan form an adhesive complex in the matrix between the neural retina and retinal pigment epithelium. Some reports have indeed shown that the insoluble-IPM is involved in the adhesion. A histochemical study using experimentally detached retinas showed that the insoluble cone matrix sheath was closely associated with both the cone photoreceptor and the apical surface of the retinal pigment epithelium, suggesting that this sheath mediated attachment (29Hollyfield J.G. Varner H.H. Rayborn M.E. Osterfeld A.M. Retina. 1989; 9: 59-68Crossref PubMed Scopus (75) Google Scholar). Insoluble IPM constituents could be found in vitreous from eyes suffering from rhegmatogenous retinal detachment (30Russell S.R. Hageman G.S. Am. J. Ophthalmol. 1997; 123: 386-391Abstract Full Text PDF PubMed Scopus (5) Google Scholar).Fig. 7 (A–C) shows the expression levels of mRNA, core protein, and sialylated O-glycan that were analyzed by using labeled cDNA-probe, O-46F, and MY-174, respectively. Successively, each expression increased in a comprehensible order during the development, because the translation processes were followed by a glycosylation process. There are some different expression patterns between mRNA and the core protein. One possibility is that the processes for transcription and translation and the glycosylation that follows are regulated in different manners in SPACR expression. Another possibility is that, at earlier developmental stages, there might be an increased turnover of the protein, because the protein levels at E15–E16 are significantly lower than the corresponding mRNA levels at the same developmental stages.Adler and Gibson (23Adler A.J. Gibson B.L. Curr. Eye Res. 1982; 2: 743-751Crossref PubMed Scopus (4) Google Scholar) showed that neural retinal adhesiveness starts at E17–E18 and that this is coincident with maturation of photoreceptor outer segments. In this study, we examined retinal adhesion using a peeling method (11Yao X.Y. Hageman G.S. Marmor M.F. Invest. Ophthalmol. Vis. Sci. 1990; 31: 2051-2058PubMed Google Scholar, 21Endo E.G. Yao X.Y. Marmor M.F. Invest. Ophthalmol. Vis. Sci. 1988; 29: 1390-1396PubMed Google Scholar, 22Marmor M.F. Prog. Retinal Res. 1993; 12: 179-204Crossref Scopus (38) Google Scholar). Although there is no adhesion at E15, we detected substantial adhesiveness at E16 by this method. The expression of SPACR in adult retina is 1.9 times the newborn level, but retinal adhesiveness of adult retina is almost equal to the newborn one. We suggest that adhesiveness does not increase after a threshold SPACR expression level is reached.Chondroitin sulfates are major constituents of the IPM (31Porrello K. LaVail M.M. Curr. Eye. Res. 1986; 5: 981-993Crossref PubMed Scopus (67) Google Scholar). Chick SPACR is not a chondroitin-type proteoglycan, because the mobility of the chick SPACR band showed no significant change after chondroitinase ABC treatment. We used the antibodies for ΔDi6S-, ΔDi4S-, and ΔDi0S-chondroitin epitopes exposed following chondroitinase ABC digestion to determine whether a chick SPACR core protein is released. That there were no specific bands for each epitope suggested that chick SPACR is not a chondroitin-type proteoglycan (data not shown), whereas Fig. 4 showed SG residues, SGD residues, and DGS residues as candidates for chondroitin sulfate attachment consensus sequences. Intracellular blocking of the attachment of chondroitin sulfate chains with xyloside prevented the secretion of proteoglycans and resulted in retinal detachment (13Lazarus H.S. Hageman G.S. Invest. Ophthalmol. Vis. Sci. 1992; 33: 364-376PubMed Google Scholar). Retinal adhesiveness was also weakened by chondroitinase ABC (11Yao X.Y. Hageman G.S. Marmor M.F. Invest. Ophthalmol. Vis. Sci. 1990; 31: 2051-2058PubMed Google Scholar). These reports suggest that chondroitin sulfate proteoglycans are involved in the adhesion. However, our report implicates a different type of molecule in this process.Our present study showed that SPACR has a pericellular distribution reminiscent of cell membranes (Fig. 1 C). To clarify whether SPACR is associated with the cell membrane, we tried a similar immunohistochemical study using O46-F and O47-F. However, these antibodies were not available for immunohistochemical study. Two SEA modules (32Bork P. Patthy L. Protein Sci. 1995; 4: 1421-1425Crossref PubMed Scopus (115) Google Scholar) corresponding to 231–348 and 728–853 in amino acid sequence positions of chick SPACR are also conserved in human and mouse SPACR (Pfam data base). The SEA module found in a number of heavily O-linked glycosylated membrane-associated adhesive proteins is considered to regulate receptor-ligand alliance by proteolytic cleavage (33Wreschner D.H. McGuckin M.A. Williams S.J. Baruch A. Yoeli M. Ziv R. Okun L. Zaretsky J. Smorodinsky N. Keydar I. Neophytou P. Stacey M. Lin H.H. Gordon S. Protein Sci. 2002; 11: 698-706Crossref PubMed Scopus (95) Google Scholar). Bishop et al. (34Bishop P.N. Boulton M. McLeod D. Stoddart R.W. Glycobiology. 1993; 3: 403-412Crossref PubMed Scopus (12) Google Scholar) showed sialylated glycans at the IPM and photoreceptor plasmalemmata by histochemical study. There is no evidence regarding the association of SPACR with the cell membrane, but SPACR is a molecule potentially associated with the cell membrane. To clarify the retinal adhesion mechanism and other biological functions of SPACR, we must establish anin vivo system.In summary, the present findings demonstrate that the MY-174 antigen at the photoreceptor layer is identical to chick SPACR. TheO-glycans on the core protein of PG-M/versican are similar to those characteristics of chick SPACR and are considered to be the antigen for MY-174. The correlation of the appearance of SPACR and the development of retinal adhesiveness suggests that SPACR may be a functional adhesive between neural retina and retinal pigment epithelium. MY-174, a monoclonal antibody, has been shown to recognize chick PG-M/versican (1Yamagata M. Shinomura T. Kimata K. Anat. Embryol. 1993; 187: 433-444Crossref PubMed Scopus (82) Google Scholar), a member of the hyaluronan-binding chondroitin sulfate proteoglycan family (2Kimata K. Oike Y. Tani K. Shinomura T. Yamagata M. Uritani M. Suzuki S. J. Biol. Chem. 1986; 261: 13517-13525Abstract Full Text PDF PubMed Google Scholar, 3Zimmermann D.R. Ruoslahti E. EMBO J. 1989; 8: 2975-2981Crossref PubMed Scopus (499) Google Scholar, 4Shinomura T. Nishida Y. Ito K. Kimata K. J. Biol. Chem. 1993; 268: 14461-14469Abstract Full Text PDF PubMed Google Scholar). In the eye, MY-174 specifically stains the photoreceptor layer (1Yamagata M. Shinomura T. Kimata K. Anat. Embryol. 1993; 187: 433-444Crossref PubMed Scopus (82) Google Scholar). However, recently we demonstrated an absence of PG-M/versican at the chick photoreceptor layer using a polyclonal antibody that recognizes all alternatively spliced forms of PG-M/versican (5Zako M. Shinomura T. Miyaishi O. Iwaki M. Kimata K. J. Neurochem. 1997; 69: 2155-2161Crossref PubMed Scopus (32) Google Scholar). These inconsistent results suggest that MY-174 does not recognize PG-M/versican but another molecule in the photoreceptor layer. The IPM (interphotoreceptormatrix),1 resides in an extracellular compartment between the outer limiting membrane of the neural retina and apical surface of the retinal pigment epithelium and is composed of proteins, glycoproteins, proteoglycans, and glycosaminoglycans (6Adler A. Severin K.M. Exp. Eye Res. 1981; 32: 755-769Crossref PubMed Scopus (91) Google Scholar, 7Adler A. Klucznik K.M. Exp. Eye Res. 1982; 34: 423-434Crossref PubMed Scopus (98) Google Scholar). A variety of important reactions relating to vision, including visual pigment chromophore exchange, metabolite trafficking, photoreceptor alignment, and membrane turnover, is thought to be mediated by the IPM (8Hageman G.S. Johnson L.V. Osborne N.N. Chader G.J. Progress in Retinal Research. Pergamon, Oxford1991: 207-249Google Scholar). However, one of the most essential functions of IPM is that of a biological glue for retinal adhesion through its viscous adhesive properties (9Zauberman H. Berman E.R. Exp. Eye Res. 1969; 8: 276-283Crossref PubMed Google Scholar, 10Zauberman H. Zinn K.M. Marmor M.F. The Retinal Pigment Epithelium. Harvard University Press, Cambridge1979: 192-204Google Scholar). Retinal adhesiveness can be weakened by treatments with enzymes such as neuraminidase, hyaluronidase, and chondroitinase ABC (11Yao X.Y. Hageman G.S. Marmor M.F. Invest. Ophthalmol. Vis. Sci. 1990; 31: 2051-2058PubMed Google Scholar, 12Yao X.Y. Hageman G.S. Marmor M.F. Invest. Ophthalmol. Vis. Sci. 1992; 33: 498-503PubMed Google Scholar). Intracellular blocking of glycosylation with xyloside also prevents the secretion of proteoglycans and results in retinal detachment (13Lazarus H.S. Hageman G.S. Invest. Ophthalmol. Vis. Sci. 1992; 33: 364-376PubMed Google Scholar). These results suggest that the proteoglycans, glycosaminoglycans, or glycoproteins of the IPM are involved in retinal adhesion. However, the specific IPM molecule that mediates adhesion has not been identified. SPACR (sialoprotein associated withcones and rods) is a hyaluronan-binding glycoprotein newly identified in adult human PBS (phosphate-buffered saline)-insoluble IPM (14Hollyfield J.G. Rayborn M.E. Landers R.A. Myers K.M. Exp. Eye Res. 1990; 51: 107-110Crossref PubMed Scopus (43) Google Scholar, 15Tien L. Rayborn M.E. Hollyfield J.G. Exp. Eye Res. 1992; 55: 297-306Crossref PubMed Scopus (32) Google Scholar, 16Acharya S. Rayborn M.E. Hollyfield J.G. Glycobiology. 1998; 8: 997-1006Crossref PubMed Scopus (35) Google Scholar, 17Acharya S. Rodriguez I.R. Moreira E.F. Midura R.J. Misono K. Todres E. Hollyfield J.G. J. Biol. Chem. 1998; 273: 31599-31606Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). This 147- to 150-kDa glycoprotein was purified by wheat germ agglutinin-affinity chromatography and characterized (17Acharya S. Rodriguez I.R. Moreira E.F. Midura R.J. Misono K. Todres E. Hollyfield J.G. J. Biol. Chem. 1998; 273: 31599-31606Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). It has been shown that (a) SPACR is heavily sialylated, (b) both N- andO-linked glycoconjugates are present in the molecule, and (c) glycoconjugates account for ∼30% of the molecular mass (17Acharya S. Rodriguez I.R. Moreira E.F. Midura R.J. Misono K. Todres E. Hollyfield J.G. J. Biol. Chem. 1998; 273: 31599-31606Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Recently, mouse SPACR was also cloned and characterized (18Lee J.W. Chen Q. Rayborn M.E. Shadrach K.G. Crabb J.W. Rodriguez I.R. Hollyfield J.G. Exp. Eye Res. 2000; 71: 341-352Crossref PubMed Scopus (14) Google Scholar). Both human and mouse SPACRs have a large central mucin-like domain flanked by consensus sites for N-linked oligosaccharide attachment, one EGF-like domain near the C-terminal, and several potential hyaluronan-binding motifs in common. Interestingly, biochemical studies showed that SPACR is a glycoprotein in human and a proteoglycan in mouse. Except for the ability of SPACR to bind hyaluronan (17Acharya S. Rodriguez I.R. Moreira E.F. Midura R.J. Misono K. Todres E. Hollyfield J.G. J. Biol. Chem. 1998; 273: 31599-31606Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), other properties or functional roles of SPACR remain unknown. In the present study, we first examined the localizations of the retinal antigen that binds to MY-174 in the photoreceptor layer. We then determined a full-length cDNA of MY-174 antigen at photoreceptor layer and showed that the antigen corresponded to chick SPACR. Finally, to investigate the biological significance of SPACR, we compared the expressions of chick SPACR and the occurrence of adhesion between neural retina and retinal pigment epithelium during development. DISCUSSIONHere we showed that a MY-174 antigen in the IPM is the ortholog of human SPACR and that there is an increase in SPACR expression during chick development. We also showed that the expression increases in parallel with retinal adhesiveness, implying that SPACR might be involved in the adhesion between neural retina and retinal pigment epithelium.We showed that sialylated O-linked glycoconjugates are involved in the epitope structure recognized by MY-174, which was developed as a monoclonal antibody to the core protein of PG-M/versican. The antigen was prepared by mild alkali treatment of the core protein of PG-M/versican, and the treatment eliminated glycoconjugates from the core protein (1Yamagata M. Shinomura T. Kimata K. Anat. Embryol. 1993; 187: 433-444Crossref PubMed Scopus (82) Google Scholar). However, when we made fusion proteins for the full length of PG-M/versican (V0) by Escherichia coli, MY-174 showed no immunoreactivity to the fusion core protein (data not shown). No immunoreactive clones could be detected by a MY-174 in screening of the chick retina cDNA library (data not shown). Acharya et al. (16Acharya S. Rayborn M.E. Hollyfield J.G. Glycobiology. 1998; 8: 997-1006Crossref PubMed Scopus (35) Google Scholar) mentioned that the major glycoconjugate on SPACR is the O-linked carbohydrate, NeuAcα2–3Galβ1–3GalNAc and that the O-linked sugar in SPACR could have a structure similar to that present in bone sialoprotein and aggrecan. At this stage we speculate that any residualO-glycans left on the core protein of PG-M/versican, specific but similar to those characteristic of SPACR, were the antigen for MY-174.Because our previous study using a polyclonal antibody that recognizes all PG-M/versican forms showed no staining in the photoreceptor layers of adult chick retinas (5Zako M. Shinomura T. Miyaishi O. Iwaki M. Kimata K. J. Neurochem. 1997; 69: 2155-2161Crossref PubMed Scopus (32) Google Scholar), we also thought it unlikely that PG-M/versican would be a target for MY-174 in retina. We have repeated this published observation with 2B1, a monoclonal antibody that recognizes all human PG-M/versican forms (25Isogai Z. Shinomura T. Yamakawa N. Takeuchi J. Tsuji T. Heinegård D. Kimata K. Cancer Res. 1996; 56: 3902-3908PubMed Google Scholar), but again no specific staining of the human photoreceptor layer could be detected (data not shown). Furthermore, no specific staining at an adult mouse photoreceptor layer could be detected using polyclonal antibodies against mouse PG-M/versican (data not shown).MY-174 staining showed resistance to a combination of neuraminidase andO-glycanase treatments around the outer segments of photoreceptor cells. Tien et al. (15Tien L. Rayborn M.E. Hollyfield J.G. Exp. Eye Res. 1992; 55: 297-306Crossref PubMed Scopus (32) Google Scholar) also reported that wheat germ agglutinin staining of SPACR is resistant to neuraminidase around the outer segments of photoreceptor cells in retinal sections. The reason for the resistance to enzymatic treatments is unclear, but the IPM seems to have different properties around inner or outer segments of photoreceptor cells.Acharya et al. (17Acharya S. Rodriguez I.R. Moreira E.F. Midura R.J. Misono K. Todres E. Hollyfield J.G. J. Biol. Chem. 1998; 273: 31599-31606Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) showed human SPACR has a functional hyaluronan-binding domain. This domain (RHAMM-type hyaluronan binding motif) corresponded to 280KEIHVLGFK288 in chick SPACR. It showed high consensus with human and mouse SPACR. This is a candidate for the RHAMM-type hyaluronan binding motif, because a single acidic group can be present in one residue inside either flanking basic residue in a B7XB RHAMM-type hyaluronan binding motif (26Yang B. Yang B.L. Savani R.C. Turley E.A. EMBO J. 1994; 13: 286-296Crossref PubMed Scopus (334) Google Scholar). Our data showed chick SPACR can bind hyaluronan. Mutagenesis studies of the putative hyaluronan binding motif will be required to establish the site of hyaluronan binding in chick SPACR. Because hyaluronan is a prominent constituent of the IPM in all species studied except mouse (27Hollyfield J.G. Rayborn M.E. Tammi R. Exp. Eye Res. 1997; 65: 603-608Crossref PubMed Scopus (18) Google Scholar, 28Hollyfield J.G. Rayborn M.E. Tammi M. Tammi R. Exp. Eye Res. 1998; 66: 241-248Crossref PubMed Scopus (44) Google Scholar), we speculate that SPACR and hyaluronan form an adhesive complex in the matrix between the neural retina and retinal pigment epithelium. Some reports have indeed shown that the insoluble-IPM is involved in the adhesion. A histochemical study using experimentally detached retinas showed that the insoluble cone matrix sheath was closely associated with both the cone photoreceptor and the apical surface of the retinal pigment epithelium, suggesting that this sheath mediated attachment (29Hollyfield J.G. Varner H.H. Rayborn M.E. Osterfeld A.M. Retina. 1989; 9: 59-68Crossref PubMed Scopus (75) Google Scholar). Insoluble IPM constituents could be found in vitreous from eyes suffering from rhegmatogenous retinal detachment (30Russell S.R. Hageman G.S. Am. J. Ophthalmol. 1997; 123: 386-391Abstract Full Text PDF PubMed Scopus (5) Google Scholar).Fig. 7 (A–C) shows the expression levels of mRNA, core protein, and sialylated O-glycan that were analyzed by using labeled cDNA-probe, O-46F, and MY-174, respectively. Successively, each expression increased in a comprehensible order during the development, because the translation processes were followed by a glycosylation process. There are some different expression patterns between mRNA and the core protein. One possibility is that the processes for transcription and translation and the glycosylation that follows are regulated in different manners in SPACR expression. Another possibility is that, at earlier developmental stages, there might be an increased turnover of the protein, because the protein levels at E15–E16 are significantly lower than the corresponding mRNA levels at the same developmental stages.Adler and Gibson (23Adler A.J. Gibson B.L. Curr. Eye Res. 1982; 2: 743-751Crossref PubMed Scopus (4) Google Scholar) showed that neural retinal adhesiveness starts at E17–E18 and that this is coincident with maturation of photoreceptor outer segments. In this study, we examined retinal adhesion using a peeling method (11Yao X.Y. Hageman G.S. Marmor M.F. Invest. Ophthalmol. Vis. Sci. 1990; 31: 2051-2058PubMed Google Scholar, 21Endo E.G. Yao X.Y. Marmor M.F. Invest. Ophthalmol. Vis. Sci. 1988; 29: 1390-1396PubMed Google Scholar, 22Marmor M.F. Prog. Retinal Res. 1993; 12: 179-204Crossref Scopus (38) Google Scholar). Although there is no adhesion at E15, we detected substantial adhesiveness at E16 by this method. The expression of SPACR in adult retina is 1.9 times the newborn level, but retinal adhesiveness of adult retina is almost equal to the newborn one. We suggest that adhesiveness does not increase after a threshold SPACR expression level is reached.Chondroitin sulfates are major constituents of the IPM (31Porrello K. LaVail M.M. Curr. Eye. Res. 1986; 5: 981-993Crossref PubMed Scopus (67) Google Scholar). Chick SPACR is not a chondroitin-type proteoglycan, because the mobility of the chick SPACR band showed no significant change after chondroitinase ABC treatment. We used the antibodies for ΔDi6S-, ΔDi4S-, and ΔDi0S-chondroitin epitopes exposed following chondroitinase ABC digestion to determine whether a chick SPACR core protein is released. That there were no specific bands for each epitope suggested that chick SPACR is not a chondroitin-type proteoglycan (data not shown), whereas Fig. 4 showed SG residues, SGD residues, and DGS residues as candidates for chondroitin sulfate attachment consensus sequences. Intracellular blocking of the attachment of chondroitin sulfate chains with xyloside prevented the secretion of proteoglycans and resulted in retinal detachment (13Lazarus H.S. Hageman G.S. Invest. Ophthalmol. Vis. Sci. 1992; 33: 364-376PubMed Google Scholar). Retinal adhesiveness was also weakened by chondroitinase ABC (11Yao X.Y. Hageman G.S. Marmor M.F. Invest. Ophthalmol. Vis. Sci. 1990; 31: 2051-2058PubMed Google Scholar). These reports suggest that chondroitin sulfate proteoglycans are involved in the adhesion. However, our report implicates a different type of molecule in this process.Our present study showed that SPACR has a pericellular distribution reminiscent of cell membranes (Fig. 1 C). To clarify whether SPACR is associated with the cell membrane, we tried a similar immunohistochemical study using O46-F and O47-F. However, these antibodies were not available for immunohistochemical study. Two SEA modules (32Bork P. Patthy L. Protein Sci. 1995; 4: 1421-1425Crossref PubMed Scopus (115) Google Scholar) corresponding to 231–348 and 728–853 in amino acid sequence positions of chick SPACR are also conserved in human and mouse SPACR (Pfam data base). The SEA module found in a number of heavily O-linked glycosylated membrane-associated adhesive proteins is considered to regulate receptor-ligand alliance by proteolytic cleavage (33Wreschner D.H. McGuckin M.A. Williams S.J. Baruch A. Yoeli M. Ziv R. Okun L. Zaretsky J. Smorodinsky N. Keydar I. Neophytou P. Stacey M. Lin H.H. Gordon S. Protein Sci. 2002; 11: 698-706Crossref PubMed Scopus (95) Google Scholar). Bishop et al. (34Bishop P.N. Boulton M. McLeod D. Stoddart R.W. Glycobiology. 1993; 3: 403-412Crossref PubMed Scopus (12) Google Scholar) showed sialylated glycans at the IPM and photoreceptor plasmalemmata by histochemical study. There is no evidence regarding the association of SPACR with the cell membrane, but SPACR is a molecule potentially associated with the cell membrane. To clarify the retinal adhesion mechanism and other biological functions of SPACR, we must establish anin vivo system.In summary, the present findings demonstrate that the MY-174 antigen at the photoreceptor layer is identical to chick SPACR. TheO-glycans on the core protein of PG-M/versican are similar to those characteristics of chick SPACR and are considered to be the antigen for MY-174. The correlation of the appearance of SPACR and the development of retinal adhesiveness suggests that SPACR may be a functional adhesive between neural retina and retinal pigment epithelium. Here we showed that a MY-174 antigen in the IPM is the ortholog of human SPACR and that there is an increase in SPACR expression during chick development. We also showed that the expression increases in parallel with retinal adhesiveness, implying that SPACR might be involved in the adhesion between neural retina and retinal pigment epithelium. We showed that sialylated O-linked glycoconjugates are involved in the epitope structure recognized by MY-174, which was developed as a monoclonal antibody to the core protein of PG-M/versican. The antigen was prepared by mild alkali treatment of the core protein of PG-M/versican, and the treatment eliminated glycoconjugates from the core protein (1Yamagata M. Shinomura T. Kimata K. Anat. Embryol. 1993; 187: 433-444Crossref PubMed Scopus (82) Google Scholar). However, when we made fusion proteins for the full length of PG-M/versican (V0) by Escherichia coli, MY-174 showed no immunoreactivity to the fusion core protein (data not shown). No immunoreactive clones could be detected by a MY-174 in screening of the chick retina cDNA library (data not shown). Acharya et al. (16Acharya S. Rayborn M.E. Hollyfield J.G. Glycobiology. 1998; 8: 997-1006Crossref PubMed Scopus (35) Google Scholar) mentioned that the major glycoconjugate on SPACR is the O-linked carbohydrate, NeuAcα2–3Galβ1–3GalNAc and that the O-linked sugar in SPACR could have a structure similar to that present in bone sialoprotein and aggrecan. At this stage we speculate that any residualO-glycans left on the core protein of PG-M/versican, specific but similar to those characteristic of SPACR, were the antigen for MY-174. Because our previous study using a polyclonal antibody that recognizes all PG-M/versican forms showed no staining in the photoreceptor layers of adult chick retinas (5Zako M. Shinomura T. Miyaishi O. Iwaki M. Kimata K. J. Neurochem. 1997; 69: 2155-2161Crossref PubMed Scopus (32) Google Scholar), we also thought it unlikely that PG-M/versican would be a target for MY-174 in retina. We have repeated this published observation with 2B1, a monoclonal antibody that recognizes all human PG-M/versican forms (25Isogai Z. Shinomura T. Yamakawa N. Takeuchi J. Tsuji T. Heinegård D. Kimata K. Cancer Res. 1996; 56: 3902-3908PubMed Google Scholar), but again no specific staining of the human photoreceptor layer could be detected (data not shown). Furthermore, no specific staining at an adult mouse photoreceptor layer could be detected using polyclonal antibodies against mouse PG-M/versican (data not shown). MY-174 staining showed resistance to a combination of neuraminidase andO-glycanase treatments around the outer segments of photoreceptor cells. Tien et al. (15Tien L. Rayborn M.E. Hollyfield J.G. Exp. Eye Res. 1992; 55: 297-306Crossref PubMed Scopus (32) Google Scholar) also reported that wheat germ agglutinin staining of SPACR is resistant to neuraminidase around the outer segments of photoreceptor cells in retinal sections. The reason for the resistance to enzymatic treatments is unclear, but the IPM seems to have different properties around inner or outer segments of photoreceptor cells. Acharya et al. (17Acharya S. Rodriguez I.R. Moreira E.F. Midura R.J. Misono K. Todres E. Hollyfield J.G. J. Biol. Chem. 1998; 273: 31599-31606Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) showed human SPACR has a functional hyaluronan-binding domain. This domain (RHAMM-type hyaluronan binding motif) corresponded to 280KEIHVLGFK288 in chick SPACR. It showed high consensus with human and mouse SPACR. This is a candidate for the RHAMM-type hyaluronan binding motif, because a single acidic group can be present in one residue inside either flanking basic residue in a B7XB RHAMM-type hyaluronan binding motif (26Yang B. Yang B.L. Savani R.C. Turley E.A. EMBO J. 1994; 13: 286-296Crossref PubMed Scopus (334) Google Scholar). Our data showed chick SPACR can bind hyaluronan. Mutagenesis studies of the putative hyaluronan binding motif will be required to establish the site of hyaluronan binding in chick SPACR. Because hyaluronan is a prominent constituent of the IPM in all species studied except mouse (27Hollyfield J.G. Rayborn M.E. Tammi R. Exp. Eye Res. 1997; 65: 603-608Crossref PubMed Scopus (18) Google Scholar, 28Hollyfield J.G. Rayborn M.E. Tammi M. Tammi R. Exp. Eye Res. 1998; 66: 241-248Crossref PubMed Scopus (44) Google Scholar), we speculate that SPACR and hyaluronan form an adhesive complex in the matrix between the neural retina and retinal pigment epithelium. Some reports have indeed shown that the insoluble-IPM is involved in the adhesion. A histochemical study using experimentally detached retinas showed that the insoluble cone matrix sheath was closely associated with both the cone photoreceptor and the apical surface of the retinal pigment epithelium, suggesting that this sheath mediated attachment (29Hollyfield J.G. Varner H.H. Rayborn M.E. Osterfeld A.M. Retina. 1989; 9: 59-68Crossref PubMed Scopus (75) Google Scholar). Insoluble IPM constituents could be found in vitreous from eyes suffering from rhegmatogenous retinal detachment (30Russell S.R. Hageman G.S. Am. J. Ophthalmol. 1997; 123: 386-391Abstract Full Text PDF PubMed Scopus (5) Google Scholar). Fig. 7 (A–C) shows the expression levels of mRNA, core protein, and sialylated O-glycan that were analyzed by using labeled cDNA-probe, O-46F, and MY-174, respectively. Successively, each expression increased in a comprehensible order during the development, because the translation processes were followed by a glycosylation process. There are some different expression patterns between mRNA and the core protein. One possibility is that the processes for transcription and translation and the glycosylation that follows are regulated in different manners in SPACR expression. Another possibility is that, at earlier developmental stages, there might be an increased turnover of the protein, because the protein levels at E15–E16 are significantly lower than the corresponding mRNA levels at the same developmental stages. Adler and Gibson (23Adler A.J. Gibson B.L. Curr. Eye Res. 1982; 2: 743-751Crossref PubMed Scopus (4) Google Scholar) showed that neural retinal adhesiveness starts at E17–E18 and that this is coincident with maturation of photoreceptor outer segments. In this study, we examined retinal adhesion using a peeling method (11Yao X.Y. Hageman G.S. Marmor M.F. Invest. Ophthalmol. Vis. Sci. 1990; 31: 2051-2058PubMed Google Scholar, 21Endo E.G. Yao X.Y. Marmor M.F. Invest. Ophthalmol. Vis. Sci. 1988; 29: 1390-1396PubMed Google Scholar, 22Marmor M.F. Prog. Retinal Res. 1993; 12: 179-204Crossref Scopus (38) Google Scholar). Although there is no adhesion at E15, we detected substantial adhesiveness at E16 by this method. The expression of SPACR in adult retina is 1.9 times the newborn level, but retinal adhesiveness of adult retina is almost equal to the newborn one. We suggest that adhesiveness does not increase after a threshold SPACR expression level is reached. Chondroitin sulfates are major constituents of the IPM (31Porrello K. LaVail M.M. Curr. Eye. Res. 1986; 5: 981-993Crossref PubMed Scopus (67) Google Scholar). Chick SPACR is not a chondroitin-type proteoglycan, because the mobility of the chick SPACR band showed no significant change after chondroitinase ABC treatment. We used the antibodies for ΔDi6S-, ΔDi4S-, and ΔDi0S-chondroitin epitopes exposed following chondroitinase ABC digestion to determine whether a chick SPACR core protein is released. That there were no specific bands for each epitope suggested that chick SPACR is not a chondroitin-type proteoglycan (data not shown), whereas Fig. 4 showed SG residues, SGD residues, and DGS residues as candidates for chondroitin sulfate attachment consensus sequences. Intracellular blocking of the attachment of chondroitin sulfate chains with xyloside prevented the secretion of proteoglycans and resulted in retinal detachment (13Lazarus H.S. Hageman G.S. Invest. Ophthalmol. Vis. Sci. 1992; 33: 364-376PubMed Google Scholar). Retinal adhesiveness was also weakened by chondroitinase ABC (11Yao X.Y. Hageman G.S. Marmor M.F. Invest. Ophthalmol. Vis. Sci. 1990; 31: 2051-2058PubMed Google Scholar). These reports suggest that chondroitin sulfate proteoglycans are involved in the adhesion. However, our report implicates a different type of molecule in this process. Our present study showed that SPACR has a pericellular distribution reminiscent of cell membranes (Fig. 1 C). To clarify whether SPACR is associated with the cell membrane, we tried a similar immunohistochemical study using O46-F and O47-F. However, these antibodies were not available for immunohistochemical study. Two SEA modules (32Bork P. Patthy L. Protein Sci. 1995; 4: 1421-1425Crossref PubMed Scopus (115) Google Scholar) corresponding to 231–348 and 728–853 in amino acid sequence positions of chick SPACR are also conserved in human and mouse SPACR (Pfam data base). The SEA module found in a number of heavily O-linked glycosylated membrane-associated adhesive proteins is considered to regulate receptor-ligand alliance by proteolytic cleavage (33Wreschner D.H. McGuckin M.A. Williams S.J. Baruch A. Yoeli M. Ziv R. Okun L. Zaretsky J. Smorodinsky N. Keydar I. Neophytou P. Stacey M. Lin H.H. Gordon S. Protein Sci. 2002; 11: 698-706Crossref PubMed Scopus (95) Google Scholar). Bishop et al. (34Bishop P.N. Boulton M. McLeod D. Stoddart R.W. Glycobiology. 1993; 3: 403-412Crossref PubMed Scopus (12) Google Scholar) showed sialylated glycans at the IPM and photoreceptor plasmalemmata by histochemical study. There is no evidence regarding the association of SPACR with the cell membrane, but SPACR is a molecule potentially associated with the cell membrane. To clarify the retinal adhesion mechanism and other biological functions of SPACR, we must establish anin vivo system. In summary, the present findings demonstrate that the MY-174 antigen at the photoreceptor layer is identical to chick SPACR. TheO-glycans on the core protein of PG-M/versican are similar to those characteristics of chick SPACR and are considered to be the antigen for MY-174. The correlation of the appearance of SPACR and the development of retinal adhesiveness suggests that SPACR may be a functional adhesive between neural retina and retinal pigment epithelium.
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