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• W2024384954 abstract "The large aggregating chondroitin sulfate proteoglycans, including aggrecan, versican (PG-M), neurocan, and brevican, are characterized by N-terminal and C-terminal globular (or selectin-like) domains known as the G1 and G3 domains, respectively. For this study, we generated a series of expression constructs containing various combinations of chicken versican/PG-M domains and a leading peptide of link protein in order to examine the roles of the G1 and G3 domains in versican function. In transfection studies, we observed that the presence of the G1 domain was sufficient to inhibit product secretion, while the G3 domain enhanced this process. We also demonstrated that the G1 domain inhibited the attachment of glycosaminoglycan chains to the core proteins, while the G3 domain enhanced this process. Further studies revealed that inhibition of secretion by G1 was mediated by its two tandem repeats, while G3's promotion of glycosaminoglycan chain attachment was apparently dependent on G3's complement-binding protein (CBP)-like motif. The modulatory effects of these two molecular domains may contribute to versican's biological activities. The large aggregating chondroitin sulfate proteoglycans, including aggrecan, versican (PG-M), neurocan, and brevican, are characterized by N-terminal and C-terminal globular (or selectin-like) domains known as the G1 and G3 domains, respectively. For this study, we generated a series of expression constructs containing various combinations of chicken versican/PG-M domains and a leading peptide of link protein in order to examine the roles of the G1 and G3 domains in versican function. In transfection studies, we observed that the presence of the G1 domain was sufficient to inhibit product secretion, while the G3 domain enhanced this process. We also demonstrated that the G1 domain inhibited the attachment of glycosaminoglycan chains to the core proteins, while the G3 domain enhanced this process. Further studies revealed that inhibition of secretion by G1 was mediated by its two tandem repeats, while G3's promotion of glycosaminoglycan chain attachment was apparently dependent on G3's complement-binding protein (CBP)-like motif. The modulatory effects of these two molecular domains may contribute to versican's biological activities. epidermal growth factor carbohydrate recognition domain complement-binding protein glycosaminoglycan chondroitin sulfate tandem repeat a miniversican construct lacking two EGF-like motifs versican G3 domain lacking two EGF-like motifs Dulbecco's modified Eagle's medium fetal bovine serum polyacrylamide gel electrophoresis polymerase chain reaction Versican, a large aggregating chondroitin sulfate proteoglycan, was initially isolated from human fibroblasts (1.Zimmermann D.R. Ruoslahti E. EMBO J. 1989; 8: 2975-2981Crossref PubMed Scopus (502) Google Scholar, 2.Naso M.F. Zimmermann D.R. Iozzo R.V. J. Biol. Chem. 1994; 269: 32999-33008Abstract Full Text PDF PubMed Google Scholar). The homologous molecule in chicken, PG-M, was first cloned from developing mesenchymes (3.Shinomura T. Nishida Y. Ito K. Kimata K. J. Biol. Chem. 1993; 268: 14461-14469Abstract Full Text PDF PubMed Google Scholar) and exists in at least four isoforms as a result of alternative splicing (4.Ito K. Shinomura T. Zako M. Ujita M. Kimata K. J. Biol. Chem. 1995; 270: 958-965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 5.Zako M. Shinomura T. Ujita M. Ito K. Kimata K. J. Biol. Chem. 1995; 270: 3914-3918Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 6.Dours-Zimmermann M.T. Zimmermann D.R. J. Biol. Chem. 1994; 269: 32992-32998Abstract Full Text PDF PubMed Google Scholar). Versican is expressed in a wide variety of tissues, including embryonic tissues (1.Zimmermann D.R. Ruoslahti E. EMBO J. 1989; 8: 2975-2981Crossref PubMed Scopus (502) Google Scholar, 7.Kimata 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, 8.Shinomura T. Jensen K.L. Yamagata M. Kimata K. Solursh M. Anat. Embryol. 1990; 181: 227-233Crossref PubMed Scopus (88) Google Scholar, 9.Yamagata M. Shinomura T. Kimata K. Anat. Embryol. 1993; 187: 433-444Crossref PubMed Scopus (82) Google Scholar), central and peripheral nervous system, the luminal surface of glandular epithelia (10.Bode-Lesniewska B. Dours-Zimmermann M.T. Odermattm B.F. Briner J. Heitz P.U. Zimmermann D.R. J. Histochem. Cytochem. 1996; 44: 303-312Crossref PubMed Scopus (163) Google Scholar), blood vessels in normal (11.Yao L.Y. Moody C. Schonherr E. Wight T.N. Sandell L.J. Matrix Biol. 1994; 14: 213-225Crossref PubMed Scopus (106) Google Scholar) and tumor (12.Paulus W. Baur I. Dours-Zimmermann M.T. Zimmerman D.R. J. Neuropathol. Exp. Neurol. 1996; 55: 528-533Crossref PubMed Scopus (95) Google Scholar) tissues, dermis, and the proliferative zone of the epidermis (13.Zimmermann D.R. Dours-Zimmermann M.T. Schubert M. Bruckner-Tuderman L. J. Cell Biol. 1994; 124: 817-825Crossref PubMed Scopus (214) Google Scholar). Its expression can be tightly regulated;e.g. during hair growth, versican expression is regulated cyclically, correlated to the hair growth cycle itself (14.du Cros D.L. LeBaron R.G. Couchman J.R. J. Invest. Dermatol. 1995; 105: 426-431Abstract Full Text PDF PubMed Scopus (112) Google Scholar). Characteristic structural features of this molecular family include a globular domain (G1) at the amino terminus directly following signal peptide, and a globular domain known as the G3 (or the selectin-like) domain at the carboxyl terminus (15.Margolis R.U. Margolis R.K. Methods Enzymol. 1994; 245: 105-126Crossref PubMed Scopus (89) Google Scholar). A large sequence named for its role in chondroitin sulfate (CS) chain attachment is situated between G1 and G3. The G1 and G3 domains are highly conserved (between species and within this proteoglycan family). The G1 domain is composed of one immunoglobulin (IgG)-like motif and two tandem repeats. This domain has the same structure as link protein, which binds hyaluronan (16.LeBaron R.G. Zimmermann D.R. Ruoslahti E. J. Biol. Chem. 1992; 267: 10003-10010Abstract Full Text PDF PubMed Google Scholar, 17.Deák F. Kiss I. Sparks K.J. Argraves W.S. Hampikian G. Goetinck P.F. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3766-3770Crossref PubMed Scopus (53) Google Scholar). The G3 domain is composed of two alternatively spliced epidermal growth factor (EGF)1-like motif(s), one lectin (also known as carbohydrate recognition domain or CRD)-like motif, one complement-binding protein (CBP)-like motif, and a short carboxyl-terminal tail. Given their ubiquitousness and their high degree of conservation, it is likely that G1 and G3 play vital roles in proteoglycan functions, but the details of these roles are only beginning to be elucidated. In the fatal chicken disorder known as nanomelia, for instance, the core protein of the proteoglycan aggrecan contains a premature stop codon N-terminal to the G3 domain. This truncated aggrecan does not contain any glycosaminoglycan (GAG) chains and is poorly secreted, suggesting that the presence of a G3 domain is crucial for proteoglycan processing and function. This study was designed to investigate the roles of G1 and G3 domains in chicken versican/PG-M synthesis and activities. We have generated a number of recombinant constructs to examine these effects. We observed that the presence of the G1 domain inhibits the attachment of glycosaminoglycan chains and secretion of the products, while the G3 domain enhances both processes. Furthermore, on the subdomain level, we demonstrated that the tandem repeats of G1 are in a large part responsible for its inhibition of versican secretion, and it is G3's CBP motif that seems to enhance GAG attachment to versican. The reverse transcription-PCR kit was fromCLONTECH. Taq DNA polymerase, T4 DNA ligase, and restriction endonucleases were from Roche Molecular Biochemicals. Bacterial growth medium was from Difco. The Prep-A-Gene DNA purification kit, prestained protein markers, and protein assay kit were from Bio-Rad. The DNA Mini-prep kit was from Bio/Can Scientific, and the DNA Midi-prep kit was from Qiagen Inc. Lipofectin, Geneticin (G418), Dulbecco's modified Eagle's medium (DMEM) growth medium, fetal bovine serum (FBS), Hanks' balanced salt solution, and trypsin/EDTA were from Life Technologies, Inc. The ECL Western blot detection kit was from Amersham Pharmacia Biotech. Horseradish peroxidase-conjugated goat anti-mouse IgG was from Sigma. 24- and 96-well tissue culture plates were from Nunc Inc. All chemicals were from Sigma. The following constructs were described previously (18.Zhang Y. Cao L. Yang B.L. Yang B.B. J. Biol. Chem. 1998; 273: 21342-21352Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 19.Zhang Y. Cao L. Yang B.L. Yang B.B. J. Biol. Chem. 1998; 273: 33054-33063Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar): miniversican, mutant miniversican (versicanΔEGF), G1, G3, mutant G3 (G3ΔEGF), miniversican lacking the G3 domain (G1CS), miniversican lacking the G1 domain (CSG3), and aggrecan G3 (aG3). Each construct contains a leading peptide (60 amino acids) originally obtained from link protein (17.Deák F. Kiss I. Sparks K.J. Argraves W.S. Hampikian G. Goetinck P.F. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3766-3770Crossref PubMed Scopus (53) Google Scholar), which contains a signal peptide and an epitope recognized by the monoclonal antibody 4B6 (20.Binette F. Cravens J. Kahoussi B. Haudenschild D.R. Goetinck P.F. J. Biol. Chem. 1994; 269: 19116-19122Abstract Full Text PDF PubMed Google Scholar). In the original cloning procedure, the G1 domain was synthesized using primers G1N and G1Ca, the CS fragment was synthesized by CSNa and CSC, and G3 was synthesized by G3Na and G3C in reverse transcription-PCR reactions. The sequences of these primers are given in Table I. The identity of each fragment was confirmed by sequencing. For this study, eight new constructs containing different combinations of motifs from versican were also produced, for a total of 16 recombinant constructs. These new constructs included G1CSCRD, G1CSCBP, G1G3, IgG, TR, CBP, CS, and CRD. Using the protocol described by us previously (18.Zhang Y. Cao L. Yang B.L. Yang B.B. J. Biol. Chem. 1998; 273: 21342-21352Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 19.Zhang Y. Cao L. Yang B.L. Yang B.B. J. Biol. Chem. 1998; 273: 33054-33063Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 21.Yang B.B. Zhang Y. Cao L. Yang B.L. Matrix Biol. 1997; 16: 537-557Crossref Google Scholar, 22.Cao L. Yang B.B. Exp. Cell Res. 1999; 246: 527-537Crossref PubMed Scopus (27) Google Scholar), we generated these constructs by subcloning and PCR methods employing the primers described in Table I.Table ISequence and restriction endonuclease sites of oligonucleotidesPrimerSequenceRestriction siteG1N5′-AAA AAG CTT ATG GTG TTA AAC ATA AAA AGC ATC ATA-3′HindIIIG1Ca5′-AAA AAA ACG CGT TTC GTA GCA GTA GGC ATC AAA CTT-3′MluIG1Cb5′-AAA GCA TGC TTC GTA GCA GTA GGC ATC-3′SphIG1Cc5′-AAA CTC GAG TTC GTA GCA GTA GGC ATC-3′XhoIIgGC5′-AAA TCT AGA ATC AAC AGC CAG CGA TAC-3′XbaITRN5′-AAA CTC GAG GGT GTA GTA TTT CAC TAT-3′XhoICSNa5′-AAA ACG CGT CGT AAA AAA ATT GTA TCA GAG CTT ACA-3′MluICSC5′-AAA CTC GAG ACT CAT TTC TGG CTC CTT TGT TTT TTC-3′XhoICSNb5′-AAA GGA TCC CGT AAA AAA ATT GTA TCA-3′BamHIG3Na5′-AAA AAA CTC GAG GGA CAG GAT CCA TGC AAA AGT AAT-3′XhoIG3C5′-AAA AAA GCA TGC GCG CCT TGA GTC CTG CCA CGT CCT-3′SphICRDN5′-AAA CTC GAG CAA GAC ACA GAG ACT-3′XhoICRDC5′-AAA TCT AGA TGT TCC TTT CTT GCA GGT-3′XbaICBPN5′-AAA CTC GAG GTT GCC TGT GGT CAA CCT-3′XhoILPN5′-AAA GAA TTC GCC GCC ACC ATG GCA AGT CTA CTC TTT-3′EcoRILP60a5′-AAA GGA TCC AGG CAG TGT GAC GTT GCC-3′BamHILP60b5′-AAA CTC GAG AGG CAG TGT GAC GTT GCC-3′XhoI Open table in a new tab Briefly, to generate the G1CSCRD construct, CRDN and CRDC were used as primers in a PCR using the G3 domain as a template. The PCR products were purified by agarose gel electrophoresis and digested withXhoI and XbaI. The digested products were purified again and used to replace the G3 fragment of the miniversican construct that had been digested with XhoI andXbaI (a cloning site in the vector) to remove the G3 fragment. In the same way, CBP fragment was synthesized with CBPN and G3C as primers and G3 domain as a template in a PCR. The PCR products were digested with XhoI and SphI and purified. The purified products were used to replace the G3 domain of the miniversican construct, which had been digested with XhoI and SphI (a cloning site in the vector). To produce the G1G3 construct, LPN and G1Cc were used as primers and the miniversican as a template in a PCR. The PCR products were digested with EcoRI and XhoI and purified. The purified products were inserted into an EcoRI- (a cloning site in the vector) and XhoI-digested miniversican construct, from which the leading peptide, the G1 domain, and CS sequence had been removed byEcoRI and XhoI digestion. In a similar way, LPN and IgGC were used as primers in a PCR using the miniversican as template. The PCR products were digested with EcoRI andXbaI, and the purified products were inserted intoEcoRI- and XbaI-digested pcDNA3 plasmid to obtain the IgG construct. To generate the TR and CBP constructs, TR was synthesized using TRN and G1Cb as primers, while CBP was synthesized using CBPN and G3C as primers. Both products were digested with XhoI andSphI. The leading peptide was synthesized with LPN and LP60b to generate an EcoRI restriction site at the amino terminus and an XhoI site at the carboxyl terminus of the leading peptide. Thus, the leading peptide, one TR fragment, and one CBP fragment were inserted into EcoRI- andSphI-digested pcDNA1 plasmid. To generate the CS construct, CS sequence was synthesized using CSNb and CSC as primers in a PCR. The PCR products were digested withBamHI and XhoI. The leading peptide was synthesized with LPN and LP60a and digested with EcoRI andBamHI. Both fragments were inserted into EcoRI- and XhoI-digested pcDNA3 plasmid. To produce the CRD construct, CRD was synthesized using CRDN and CRDC as primers in a PCR. The PCR products were digested withXhoI and XbaI and purified. The purified products were inserted into an XhoI- and XbaI-digested CBP construct, in which the XhoI site was situated between the leading peptide and the CBP fragment, while the XbaI site was located 3′ of the SphI site. COS-7 cells, astrocytoma cell line U87, or NIH3T3 fibroblasts, cultured in six-well plates at a density of 1.5 × 105 cells/well, were allowed to attach and grow overnight in DMEM supplemented with 5% FBS. The following day, Lipofectin (10 μl) was incubated with plasmid DNA (2 μg) for 15 min in 200 μl of DMEM followed by the addition of 800 μl of DMEM. The Lipofectin-DNA mixture was added to washed cultures, and the cultures were incubated in an incubator at 37 °C. After 5 h, the DNA/Lipofectin mixture was replaced with 2 ml of DMEM supplemented with 5% FBS. The culture medium and cell lysate were harvested separately after 3 days, and samples were frozen at −20 °C until analysis. Cell lysate and growth medium that contained recombinant gene products were harvested from transfected cultures and subjected to SDS-PAGE. Immunoblotting was carried out as described previously (18.Zhang Y. Cao L. Yang B.L. Yang B.B. J. Biol. Chem. 1998; 273: 21342-21352Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 19.Zhang Y. Cao L. Yang B.L. Yang B.B. J. Biol. Chem. 1998; 273: 33054-33063Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). All products were detected using the 4B6 antibody. Densitometer readings were used to estimate relative protein concentration from the intensity of the chemiluminescent signal, according to the manufacturer's instructions (Molecular Dynamics Inc., Sunnyvale, CA). Results were reported below each Western blot analyzed. This study is designed to investigate the roles of the G1 domain and the G3 domain in versican biosynthesis and secretion. We have previously observed that the versican signal peptide is not sufficient for secretion of the G1 domain (data not shown). Replacing the native versican signal peptide with the leading peptide of link protein resulted in secretion of G1. For reasons of consistency, we have substituted the link protein leading peptide for the endogenous signal peptide in every construct used in this study (Fig.1). To investigate the role of the G1 domain of versican in product secretion, the CS and G1 constructs (Fig. 1) were expressed in COS-7 cells as described under “Experimental Procedures.” Growth medium was collected and analyzed on Western blot probed with the monoclonal antibody 4B6, which recognizes an epitope within the leading peptide. G1 and CS products were produced and secreted to the media as expected (Fig. 2 A), except that CS expression was significantly higher than that of G1. Using these constructs, we then compared glycosaminoglycan chain attachment to the CS-bearing sequence of the core protein. COS-7 cells were transfected with G1CS and CS constructs, and the growth medium was analyzed on Western blot. Both constructs produced a characteristic proteoglycan smear after immunoblotting (Fig. 2 B), indicating that the core proteins of the recombinant constructs were modified by GAG chain attachment. However, the CS construct exhibited higher levels of GAG modification than did the G1CS construct. In view of the reduced secretion of the G1 product, we sought to identify specific motif(s) in the G1 domain responsible for inhibition of secretion. We thus generated an IgG construct and a TR construct containing two tandem repeats (Fig. 1) and expressed them in COS-7 cells. Cell lysate and culture medium were harvested and analyzed on Western blot. Both constructs were well expressed, and significant amounts of products were found in the cell lysate (Fig.3). However, while the product of the IgG construct was secreted into the medium at high levels, the product of the TR construct was not observed in the media at all. Prolonged exposure of the Western blot revealed two weak bands in the lane containing the greatest amount of loaded media, suggesting glycosylation of the products. We further investigated the time course of biosynthesis and secretion of TR products. COS-7 cells were transfected with the TR construct and cultured for 2, 3, or 5 days after transfection. Western blot analysis showed that TR products were expressed after only 2 days of transfection, but secretion was weak even 5 days after transfection (Fig.4).Figure 4A time course of TR product secretion.COS-7 cells were transiently transfected with the TR construct and cultures were maintained in DMEM, 5% FBS for different periods as indicated in the figure (2, 3, or 5 days). Cell lysate and culture media were harvested, and 10 or 20 μl was analyzed on Western blot probed with 4B6. Secretion of TR products to the culture media was readily detected after 3 days.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The work done in this laboratory and others has suggested that the G3 domain has important physiological functions (18.Zhang Y. Cao L. Yang B.L. Yang B.B. J. Biol. Chem. 1998; 273: 21342-21352Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 19.Zhang Y. Cao L. Yang B.L. Yang B.B. J. Biol. Chem. 1998; 273: 33054-33063Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar,23.Li H. Schwartz N.B. Vertel B.M. J. Biol. Chem. 1993; 268: 23504-23511Abstract Full Text PDF PubMed Google Scholar, 24.Vertel B.M. Walters L.M. Grier B. Maine N. Goetinck P.F. J. Cell Sci. 1993; 104: 939-948Crossref PubMed Google Scholar, 25.Luo W. Kuwada T.S. Chandrasekaran L. Zheng J. Tanzer M. J. Biol. Chem. 1996; 271: 16447-16450Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 26.Yang B. Yang B.L. Goetinck P.F. Anal. Biochem. 1994; 228: 299-306Crossref Scopus (44) Google Scholar). This led us to investigate the role of the versican G3 domain in biosynthesis and product secretion. We transfected COS-7 cells with the G1 and G3 constructs. Gene products were analyzed as above. Transfected cells secreted significantly greater amounts of G3 than G1 (Fig. 5 A). Cell lysate obtained from G3-transfected cells also contained a higher level of the G3 product. A similar level of secretion was observed in cells transfected with the CSG3 construct, implying that the G3 domain of the recombinant gene plays an important role in biosynthesis and secretion of proteoglycans. Further confirmation came from comparison of the G1CS and CSG3 constructs. COS-7 cells transfected with CSG3 construct produced significantly higher levels of products, and these products were secreted at greater levels than were G1CS gene products (Fig.5 B). This experiment also showed that the levels of GAG chain modification on the G1CS and CSG3 core proteins were very different. The sizes of the core proteins of G1CS and CSG3 constructs are similar, so the fact that the smear bands in CSG3-transfected cells (Fig. 5 B, medium) were much more intense than in G1CS-transfected cells suggested that there were more GAG chains attached to the CS sequence of CSG3 construct than to G1CS construct and that more products of CSG3 were secreted to the medium than G1CS products. These results suggested again that the G3 domain promoted GAG attachment. In view of the significant but distinct effects of these two domains, we generated a G1G3 construct to examine interdomain interaction. Its expression and secretion were analyzed as above. More G1G3 product was observed in the cell lysate than in the culture medium (Fig.6). This suggested that secretion of the G3 product is inhibited by G1 when the two domains are physically linked. The G3 domain is composed of two EGF-like motifs, a CRD-like motif and a CBP-like motif. When the two EGF-like motifs were deleted from the G3 domain, the resulting G3ΔEGF construct was synthesized by transfected cells and secreted to the medium at the same levels as the G3 product itself (Fig. 7 A). Aggrecan G3 domain, which does not contain any EGF-like motifs, showed similar behavior. Consistent results were also observed when the two EGF-like motifs were removed from the miniversican construct. Miniversican and versicanΔEGF showed comparable levels of biosynthesis, GAG chain attachment, and product secretion (Fig. 7 B). When CRD and CBP constructs were transfected into COS-7 cells, their products were secreted to the culture media (Fig.8 A). However, CBP was synthesized and secreted in greater amounts than was CRD. Concomitant with this, CBP products showed higher levels of modification by glycosaminoglycan chains and product secretion, resulting in more intense smears on the blot. To test the effects of the CRD and CBP motifs on GAG chain attachment to the CS sequence, COS-7 cells were transfected with constructs lacking EGF-like motif plus one of these two motifs (G1CSCRD and G1CSCBP constructs). Three days after transfection, culture media and cell lysate were prepared and analyzed on Western blot. The results revealed that GAG chains attached to the core proteins of both constructs; however, the levels of GAG chain modification were significantly higher on the G1CSCBP products than on G1CSCRD products (Fig. 8 B). The synthesis and secretion of the G1 and G3 constructs were also examined in cell types other than COS-7 cells. Both constructs were transiently expressed in astrocytoma cell line U87 and NIH3T3 fibroblasts. Analysis of culture medium and cell lysate from transfected cultures indicated that the products of G1 were poorly secreted as compared with G3 products, although both constructs were well synthesized (Fig. 9). Versican, also known as PG-M, is a member of the large aggregating chondroitin sulfate proteoglycan family (1.Zimmermann D.R. Ruoslahti E. EMBO J. 1989; 8: 2975-2981Crossref PubMed Scopus (502) Google Scholar, 3.Shinomura T. Nishida Y. Ito K. Kimata K. J. Biol. Chem. 1993; 268: 14461-14469Abstract Full Text PDF PubMed Google Scholar). This family also includes aggrecan (23.Li H. Schwartz N.B. Vertel B.M. J. Biol. Chem. 1993; 268: 23504-23511Abstract Full Text PDF PubMed Google Scholar), neurocan (27.Rauch U. Karthikeyan L. Maurel P. Margolis R.K. Margolis R.U. J. Biol. Chem. 1992; 267: 19536-19547Abstract Full Text PDF PubMed Google Scholar), and brevican (28.Yamada H. Watanabe K. Shimonaka M. Yamaguchi Y. J. Biol. Chem. 1994; 269: 10119-10126Abstract Full Text PDF PubMed Google Scholar). We have previously demonstrated that versican reduced cell adhesion (29.Yang B.L. Zhang Y. Cao L. Yang B.B. J. Cell. Biochem. 1999; 72: 210-220Crossref PubMed Scopus (92) Google Scholar, 30.Zhang Y. Cao L. Kiani C. Yang B.L. Hu W. Yang B.B. J. Cell. Biochem. 1999; 73: 445-457Crossref PubMed Scopus (65) Google Scholar) and stimulated cell migration (31.Ang L.C. Zhang Y. Cao L. Yang B.L. Young B. Kiani C. Lee V. Allan K. Yang B.B. J. Neuropathol. Exp. Neurol. 1999; 58: 597-605Crossref PubMed Scopus (79) Google Scholar) via the G1 domain and promoted cell proliferation (18.Zhang Y. Cao L. Yang B.L. Yang B.B. J. Biol. Chem. 1998; 273: 21342-21352Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) and inhibited cell differentiation (19.Zhang Y. Cao L. Yang B.L. Yang B.B. J. Biol. Chem. 1998; 273: 33054-33063Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) via the G3 domain. This study was motivated by reports indicating that the G1 and G3 domains of aggrecan are also important in the synthesis and secretion of this proteoglycan (25.Luo W. Kuwada T.S. Chandrasekaran L. Zheng J. Tanzer M. J. Biol. Chem. 1996; 271: 16447-16450Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 32.Zheng J. Luo W. Tanzer M.L. J. Biol. Chem. 1998; 273: 12999-13006Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The roles of versican G1 and G3 domains are not yet defined, but since versican expression is highly regulated during tissue development and maturation, we investigated the possibility that the G1 and G3 domains of versican play a part in this regulation. To perform this study, we found it necessary to substitute the link protein leading peptide for the versican signal peptide, to permit maximal secretion of secretable products, since preliminary studies showed that constructs containing the versican signal peptide were retained within the cells. The presence of this peptide also allowed detection of all constructs using a single antibody (4B6, which recognizes an epitope in the link protein leading peptide). We observed that products of the G1 and G3 constructs containing the link protein leading peptide were able to secrete to the culture media. However, comparison of the levels of G1 and G3 products in the culture medium and in the cell lysate indicated that product secretion of G1 was inhibited. This result was confirmed in studies comparing a miniversican construct with a miniversican construct lacking the G1 domain (CSG3) and comparing a G1CS construct with a CS construct. In all cases, expression and secretion of the G1-containing constructs were reduced compared with controls. The inhibition of G1 secretion and promotion of G3 secretion were also observed in NIH3T3 fibroblasts and astrocytoma cell line U87. Given that the CS sequence by itself was well synthesized, modified by GAG chains, and secreted, the addition of G1 to this sequence had dramatic inhibitory effects on its synthesis, GAG chain attachment, and product secretion. On the other hand, G3's stimulatory effects on synthesis and secretion were not evident in the CSG3 construct, since CS was already synthesized and secreted at high levels. The effects of G3 were seen by contrasting the G1CS construct and G1CSG3 (miniversican) construct, since the inclusion of G3 led to increased GAG chain attachment and secretion. Although versican is expressed in a variety of tissues, its expression is strictly regulated. For example, versican expression is highly elevated in the early stages of development (7.Kimata 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, 8.Shinomura T. Jensen K.L. Yamagata M. Kimata K. Solursh M. Anat. Embryol. 1990; 181: 227-233Crossref PubMed Scopus (88) Google Scholar). As a result, developing mesenchymes contain high levels of versican. In these stages, versican may enhance cell proliferation and the growth of limb buds as we have demonstrated that versican stimulated growth of NIH3T3 fibroblast (18.Zhang Y. Cao L. Yang B.L. Yang B.B. J. Biol. Chem. 1998; 273: 21342-21352Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) and chicken chondrocytes (30.Zhang Y. Cao L. Kiani C. Yang B.L. Hu W. Yang B.B. J. Cell. Biochem. 1999; 73: 445-457Crossref PubMed Scopus (65) Google Scholar). However, during maturation, versican expression is significantly down-regulated (13.Zimmermann D.R. Dours-Zimmermann M.T. Schubert M. Bruckner-Tuderman L. J. Cell Biol. 1994; 124: 817-825Crossref PubMed Scopus (214) Google Scholar), and the mature cartilage contains small amounts of versican that are only detected with reverse transcription-PCR techniques (33.Grover J. Roughley P. Biochem. J. 1993; 291: 361-367Crossref PubMed Scopus (61) Google Scholar). Versican expression may be down-regulated to allow cell differentiation. We earlier demonstrated that versican inhibits mesenchymal chondrogenesis (19.Zhang Y. Cao L. Yang B.L. Yang B.B. J. Biol. Chem. 1998; 273: 33054-33063Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), and versican expression has been shown to be up- and down-regulated during hair growth cycles (14.du Cros D.L. LeBaron R.G. Couchman J.R. J. Invest. Dermatol. 1995; 105: 426-431Abstract Full Text PDF PubMed Scopus (112) Google Scholar); versican expression is up-regulated during hair growth and down-regulated just before hair enters the next cycle (14.du Cros D.L. LeBaron R.G. Couchman J.R. J. Invest. Dermatol. 1995; 105: 426-431Abstract Full Text PDF PubMed Scopus (112) Google Scholar). We speculate that versican is subjected to dual modes of regulation, mediated via G1 and G3 domains. Cells may have developed pathways that allow versican up- or down-regulation (via G3 or G1, respectively) during various stages, to promote or inhibit cell growth and differentiation. In tissues where moderate levels of versican are required, both G1- and G3-regulating pathways would function to permit moderate expression and maintain a balance in versican expression. The significance of the effects of G1 and G3 on GAG chain attachment are not immediately clear. Two lines of evidence suggest that regulation of GAG chain attachment may be important during tissue development. It has been reported that attachment of GAG chains changes during tissue growth, and the types and numbers of GAG chains vary during different growth stages (34.Block J.A. Inerot S.E. Gitelis S. Kimura J.H. Am. J. Bone Joint Surg. 1991; 73: 647-658Crossref PubMed Scopus (48) Google Scholar). Furthermore, the GAG chains of versican have been reported to be involved in reducing cell adhesion (35.Yamagata M. Suzuki S. Akiyama S.K. Yamada K.M. Kimata K. J. Biol. Chem. 1989; 264: 8012-8018Abstract Full Text PDF PubMed Google Scholar). Since a decrease in cell adhesion is required for cell proliferation, cell migration, and tissue outgrowth, it seems that G3's promotion of versican synthesis, GAG chain attachment, and product secretion are relevant to tissue growth. G1's ability to inhibit these processes may be required for cell differentiation and tissue maturation. Although the concentration of versican may change to meet the requirements for tissue growth and maturation, it is not known if G1 and G3 are directly involved in the synthesis and attachment of GAG chains to versican core protein during tissue growth and differentiation. In vitro observations could well differ from in vivo effects. The significance of G1-mediated inhibition and G3-mediated enhancement of GAG chain attachment in cell culture awaits confirmation in tissue studies. We thank Dr. Paul F. Goetinck for the 4B6 monoclonal antibody." @default.
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• W2024384954 title "Tandem Repeats Are Involved in G1 Domain Inhibition of Versican Expression and Secretion and the G3 Domain Enhances Glycosaminoglycan Modification and Product Secretion via the Complement-binding Protein-like Motif" @default.
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