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• W2002167434 abstract "Aggrecan is the major proteoglycan in the extracellular matrix of cartilage. A notable exception is nanomelic cartilage, which lacks aggrecan in its matrix. The example of nanomelia and other evidence leads us to believe that the G3 domain plays an important role in aggrecan processing, and it has indeed been confirmed that G3 allows glycosaminoglycan (GAG) chain attachment and product secretion. However, it is not clear how G3, which contains at least a carbohydrate recognition domain (CRD) and a complement binding protein (CBP) motif, plays these two functional roles. The present study was designed to dissect the mechanisms of this phenomenon and specially 1) to determine the effects of various cysteine residues in GAG modification and product secretion as well as 2) to investigate which of the two processing events is the critical step in the product processing. Our studies demonstrated that removal of the two amino-terminal cysteines in the CRD motif and the single cysteine in the amino terminus of CBP inhibited secretion of CRD and CBP. Use of the double mutant CRD construct also allowed us to observe a deviation from the usual strict coupling of GAG modification and product secretion steps. The presence of a small chondroitin sulfate fragment overcame the secretion-inhibitory effects once the small chondroitin sulfate fragment was modified by GAG. Aggrecan is the major proteoglycan in the extracellular matrix of cartilage. A notable exception is nanomelic cartilage, which lacks aggrecan in its matrix. The example of nanomelia and other evidence leads us to believe that the G3 domain plays an important role in aggrecan processing, and it has indeed been confirmed that G3 allows glycosaminoglycan (GAG) chain attachment and product secretion. However, it is not clear how G3, which contains at least a carbohydrate recognition domain (CRD) and a complement binding protein (CBP) motif, plays these two functional roles. The present study was designed to dissect the mechanisms of this phenomenon and specially 1) to determine the effects of various cysteine residues in GAG modification and product secretion as well as 2) to investigate which of the two processing events is the critical step in the product processing. Our studies demonstrated that removal of the two amino-terminal cysteines in the CRD motif and the single cysteine in the amino terminus of CBP inhibited secretion of CRD and CBP. Use of the double mutant CRD construct also allowed us to observe a deviation from the usual strict coupling of GAG modification and product secretion steps. The presence of a small chondroitin sulfate fragment overcame the secretion-inhibitory effects once the small chondroitin sulfate fragment was modified by GAG. glycosaminoglycan the globular domain in the carboxyl terminus of aggrecan or selectin-like domain carbohydrate recognition domain complement binding protein chondroitin sulfate (chain attachment sequence) a small CS fragment containing amino acids 1274–1362 of chicken aggrecan and 8 consensus sequences for GAG chain modification Dulbecco's modified Eagle's medium Proteoglycans are a family of glycoconjugates with a central core protein to which the glycosaminoglycan (GAG)1 side chain(s) is covalently linked post-translationally (1Wight T.N. Heinegård D.K. Hascall V.C. Hay E.D. Cell Biology of Extracellular Matrix. Plenum Press, New York1991: 45-78Crossref Google Scholar). The functions of proteoglycans are due in large part to their GAG chains (2Watanabe H. Kimata K. Line S. Strong D. Gao L-Y. Kozak C.A. Yamada Y. Nat. Genet. 1994; 7: 154-157Crossref PubMed Scopus (209) Google Scholar, 3Goetinck P.F. Bode H. Current Topics of Developmental Biology. Academic Press, Inc., New York1991: 111-131Google Scholar). These GAG chains are acidic and participate in a wide variety of interactions with other matrix macromolecules, cations, and water (4Toole B.P. Cell Biology of Extracellular Matrix. Plenum Press, New York1991: 305-342Crossref Google Scholar, 5Vilim V. Fosang A.J. Biochem. J. 1994; 304: 887-894Crossref PubMed Scopus (32) Google Scholar). They can sequester a variety of extracellular proteins at cell surfaces. In cartilage, the molecules that make up the extracellular matrix include proteoglycans, hyaluronan, type II collagen, and glycoproteins. Aggrecan is the major structural proteoglycan in cartilage and is responsible for its resilience and load-bearing properties. Loss of aggrecan is a major feature of cartilage degradation associated with arthritis (6Lohmander L.S. Dahlberg L. Ryd L. Heinegard D. Arthritis Rheum. 1989; 32: 1434-1442Crossref PubMed Scopus (171) Google Scholar, 7Lohmander L.S. Hoerner L.A. Dahlberg L. Roos H. Bjornsson S. Lark M.W. J. Rheumatol. 1993; 20: 1362-1368PubMed Google Scholar). The core protein of aggrecan is composed of three globular domains (G1, G2, and G3) with a large extended region between G2 and G3 for GAG chain attachment, CS (1Wight T.N. Heinegård D.K. Hascall V.C. Hay E.D. Cell Biology of Extracellular Matrix. Plenum Press, New York1991: 45-78Crossref Google Scholar, 8Margolis R.U. Margolis R.K. Methods Enzymol. 1994; 245: 105-126Crossref PubMed Scopus (89) Google Scholar, 9Chandrasekaran L. Tanzer M.L. Biochem. J. 1992; 288: 903-910Crossref PubMed Scopus (30) Google Scholar). G1 comprises the amino terminus of the core protein. This domain has the same structural motif as link protein (10Deak 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). G2 is homologous to the tandem repeats of G1 and of link protein. G3, which makes up the carboxyl terminus of the core protein, is composed of folded modules including alternatively spliced epidermal growth factor-like motifs, a carbohydrate recognition domain (CRD), a complement binding protein (CBP)-like domain, and a short carboxyl-terminal tail (11Sai S. Tanaka T. Kosher R.A. Tanzer M.L. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5081-5085Crossref PubMed Scopus (61) Google Scholar). The G3 domain seems to be important in aggrecan processing. This was initially observed in chicken nanomelia, in which a point mutation produces a premature stop codon on the amino-terminal side of G3 (12Li H. Schwartz N.B. Vertel B.M. J. Biol. Chem. 1993; 268: 23504-23511Abstract Full Text PDF PubMed Google Scholar). This truncated core protein generates a lethal phenotype in homozygous form (failure of chondrogenesis and osteogenesis) and dwarfism in heterozygous form (12Li H. Schwartz N.B. Vertel B.M. J. Biol. Chem. 1993; 268: 23504-23511Abstract Full Text PDF PubMed Google Scholar). In the nanomelic mutation, the cartilage matrix lacks aggrecan. The mutation seems to inhibit aggrecan secretion to the matrix, and no modification by GAG chains occurs (13Vertel B.M. Walters L.M. Grier B. Maine N. Goetinck P.F. J. Cell Sci. 1993; 104: 939-948Crossref PubMed Google Scholar,14Vertel B.M. Walters L.M. Flay N. Kearns A.E. Schwartz N.B. J. Biol. Chem. 1993; 268: 11105-11112Abstract Full Text PDF PubMed Google Scholar). It has been proposed that G3 may regulate the attachment of GAG chains and affect the secretion of aggrecan, and it was later demonstrated that G3 is important in product secretion (15Vertel B.M. Grier B.L. Li H. Schwartz N.B. Biochem. J. 1994; 301: 211-216Crossref PubMed Scopus (44) Google Scholar). Recently, data from independent studies indicated that the G3 domain of aggrecan and also versican plays an important role in GAG chain attachment and product secretion (16Luo W. Kuwada T.S. Chandrasekaran L. Zheng J. Tanzer M.L. J. Biol. Chem. 1996; 271: 16447-16450Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 17Zheng J. Luo W. Tanzer M.L. J. Biol. Chem. 1998; 273: 12999-13006Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 18Day J.M. Murdoch A.D. Hardingham T.E. J. Biol. Chem. 1999; 274: 38107-38111Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 19Domowicz M.S. Pirok III, E.W. Novak T.E. Schwartz N.B. J. Biol. Chem. 2000; 275: 35098-35105Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 20Yang B.L. Cao L. Kiani C. Lee V. Zhang Y. Adams M.E. Yang B.B. J. Biol. Chem. 2000; 275: 21255-21261Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 21Kiani C. Lee V. Cao L. Chen L. Wu Y. Zhang Y. Adams M.E. Yang B.B. Biochem. J. 2001; 354: 199-207Crossref PubMed Scopus (33) Google Scholar). The question remains, is it possible to distinguish which step is more important in the process? The present study was designed to dissect the mechanisms governing the effects of the G3 domain in GAG chain attachment and product secretion. We generated a large number of deletion mutants of the G3 domain to investigate whether G3 conformation, through the formation of cysteine-mediated disulfide bonds, was involved. We determined that cysteines present in G3 do indeed play a role in product secretion. We also used mutants to distinguish G3 structural elements that allow product secretion without enhancing GAG chain attachment and vice versa. Taq DNA polymerase, T4 DNA ligase, and restriction endonucleases were purchased from Roche Molecular Biochemicals or Invitrogen. Bacterial growth medium was from Difco. Prestained protein markers were from New England Biolabs. Lipofectin, Geneticin (G418), Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, Hanks' balanced salt solution, and trypsin/EDTA were from Invitrogen. The ECL Western blot detection kit was from Amersham Biosciences, Inc. Horseradish peroxidase-conjugated goat anti-mouse IgG was from Sigma. The DNA mini-prep kits and midi-prep kits were from Qiagen Inc. Tissue culture plates (12-well, 6-well, and 100 mm) were from Nunc. Inc. All chemicals were from Sigma. COS-7 cells, from American Type Culture Collection, were cultured in DMEM supplemented with 5% fetal bovine serum at 37 °C in a humidified incubator containing 5% CO2. In this study, a total of 32 recombinant constructs were used. Of these, 22 (including the G3 construct) were generated based on an aggrecan G3 construct, which was cloned and expressed in our previous studies (22Yang B.B. Zhang Y. Cao L. Yang B.L. Matrix Biol. 1997; 16: 537-557Crossref Google Scholar,23Cao L. Zhang Y. Yang B.B. Matrix Biol. 1998; 17: 379-392Crossref PubMed Scopus (16) Google Scholar). This construct contains the leading peptide, which is composed of a link protein signal peptide and an epitope recognized by the monoclonal antibody 4B6 (24Binette F. Cravens J. Kahoussi B. Haudenschild D.R. Goetinck P.F. J. Biol. Chem. 1994; 269: 19116-19122Abstract Full Text PDF PubMed Google Scholar), and the aggrecan G3 domain with the carboxyl tail in pcDNA3. The leading peptide and G3 domain are linked by a XhoI restriction endonuclease site, whereas the tail and the vector are linked by an XbaI site. Individual mutants were generated using PCR. All names of the primers are given in Fig. 1, and the oligonucleotide sequences are listed in TableI. The location of primers and the presence or absence of cysteines are also given in Fig. 1. All 5′ primers contained the XhoI restriction site, and 3′ primers contained the XbaI restriction site, allowing insertion of the PCR-derived fragments into XhoI- andXbaI-digested G3 construct. Thus, the G3 domain of the G3 construct was replaced with the PCR-generated fragments. Twenty such constructs are given in Fig. 1, whereas the constructs G3 and CRDCBP (generated by primers CRDNXhoI and CBPCXbaI) are given in Fig. 5.Table ISequence and restriction endonuclease sites for oligonucleotidesPrimerSequenceCRCmuC1XhoI5′-GGG CTC GAG TGG GAG GAA GGC TGG ATC AAGCRDmuC2XhoI5′-GGG CTC GAG TGT GAG GAA GGC TGG ATG AAG TTC CAG GGC CAC GGCCRDmuC1+2XhoI5′-GGG CTC GAG ATC AAG TTC CAG GGC CAC GGCCRDmuC3XhoI5′-GGG CTC GAG ATG GAC GCA GAG TCC AGG GGCCRDmuC4XbaI5′-GGG TCT AGA ATG CCA GAT CAT CAC AAC GCC GTCCRDmuC5XbaI5′-GGG TCT AGA CTT GCA CGT GAA AGG CAG GTG ATA GTT GCC GGGCRDmuC6XbaI5′-GGG TCT AGA CTT GCC CGT GAA AGG CAG GTGCRDmuC5+6XbaI5′-GGG TCT AGA AGG CAG GTG ATA GTT GCC GGGCRDNXhoI5′-AAA AAA CTC GAG GAC CTG GCA AAC TGT GAG GAACRDCXbaI5′-AAA AAA TCT AGA GTG ATG GTG ATG GTG ATG AAC TGT TTC CTT CTT GCACBPmuC1XhoI5′-GGG CTC GAG GCC TGG GGG GAC CCA CCT GTACBPmuC3XbaI5′-GGG TCT AGA GCA GGA TAT CCG CGG TTC CTC CCA GTG CCC GTT GGG TTG CCACBPmuC4XbaI5′-GGG TCT AGA CCA GGA TAT CCG CGG TTC CTCCBPmuC3+4XbaI5′-GGG TCT AGA CCA GTG CCC GTT GGG TTG CCACBPNXhoI5′-AAA AAA CTC GAG GTT GCC TGT GGG GAC CCA CCTCBPCXbaI5′-AAA TCT AGA GTT TGT GCA GGA TAT CCGG3CXbaI5′-AAA TCT AGA GTG ATG GTG ATG GTG ATG ATG GGT GGG TCT GTG CAC Open table in a new tab Figure 5CBP alone does not account for G3 function inGAG chain attachment.A, five constructs, G1CSDG3, G1CSDCRDCBP, G1CSDCRD, G1CSDCBPtail, and G1CSD, were generated to investigate the effects of CRD and CBP on GAG chain attachment. Cell lysate and culture medium from COS-7 cells transfected with these 5 constructs were analyzed on Western blot probed with 4B6. All constructs were well expressed; however, the product of G1CSD was not modified by GAG chains and not secreted, whereas the product of G1CSDCBPtail was poorly modified by GAG chains and poorly secreted (B). The constructs CRDCBP and G3 (see panel A) were well expressed, and the products were secreted (C). D, the apparent molecular masses of the products in cell lysate (lys) and culture medium (med) were much greater than the calculated molecular mass (cal) of the recombinant constructs. E, after treatment with chondroitinase ABC, the secreted products migrated as single bands on Western blot probed with 4B6. The numbers above the schematic correspond to nucleotides in the sequence of full-length aggrecan. LP, leading peptide obtained from link protein; IgG, immunoglobular-like domain; TR, tandem repeat; control, products of G1CSDG3 without chondroitinase ABC treatment.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The remaining 10 constructs were based on a G1CSDG3 construct originally described by us (21Kiani C. Lee V. Cao L. Chen L. Wu Y. Zhang Y. Adams M.E. Yang B.B. Biochem. J. 2001; 354: 199-207Crossref PubMed Scopus (33) Google Scholar). Also known as “mini-aggrecan,” this construct contains the leading peptide (for antibody recognition), the aggrecan G1 domain, a small CS fragment (CSD) containing amino acids 1274–1362 of chicken aggrecan and 8 consensus sequences for GAG chain modification (9Chandrasekaran L. Tanzer M.L. Biochem. J. 1992; 288: 903-910Crossref PubMed Scopus (30) Google Scholar), the aggrecan G3 domain, and a short carboxyl-terminal tail. To create the mutants, various domains were deleted (Fig. 5) or swapped with CRD and CBP domains containing mutated cysteines (Fig. 6). Because the G3 domain in G1CSDG3 contains a 5′XhoI site and a 3′ XbaI site, the CRDCBP fragment was synthesized with the primers CRDNXhoI (containing theXhoI site) and CBPCXbaI (containing the XbaI site). All other fragments were obtained from some of the 20 constructs described above (also containing XhoI at 5′ andXbaI at 3′). DNA was amplified in PCR using pairs of appropriate primers. The reaction mixture (total volume, 100 μl) contained 200 μm dNTPs, 0.2 μg of each primer, 50 ng of template DNA, 5 units of Taq DNA polymerase, and the Mg2+-containing buffer (Roche Molecular Biochemicals). The reactions were carried out at 94 °C for 5 min for 1 cycle, 94 °C (60 s), 55 °C (60 s), and 72 °C (60 s) for 25 cycles, and a final extension at 72 °C for 10 min. DNA products were purified and then doubly digested with two appropriate restriction endonucleases. The DNA was ligated into the appropriate linearized plasmids (e.g. pcDNA3). A ligation mixture typically contained 2 μl of 5× ligation buffer, 1 μl of T4 DNA ligase, 3 μl of plasmid vector (50 ng), and 4 μl of insert fragment (150 ng). The ligation reaction was carried out at 16 °C overnight. Five μl of the ligation mixture was used for heat shock transformation of competent Escherichia coli strain DH5α. Bacteria were then transferred to 500 μl of SOC medium (0.5% yeast extract, 2% tryptone, 10 mm NaCl, 2.5 mm KCl, 20 mm MgSO4, 20 mm MgCl2, 20 mm glucose, pH 7.0), agitated at 225 rpm for 1 h at 37 °C, spread onto LB agar plates containing 100 μg ampicillin/ml, and cultured at 37 °C overnight. The identities of all new constructs were confirmed by DNA sequencing performed by the Core Molecular Biology Laboratory at York University (Toronto, Ontario). The results were then compared with the published sequences (9Chandrasekaran L. Tanzer M.L. Biochem. J. 1992; 288: 903-910Crossref PubMed Scopus (30) Google Scholar, 12Li H. Schwartz N.B. Vertel B.M. J. Biol. Chem. 1993; 268: 23504-23511Abstract Full Text PDF PubMed Google Scholar). To analyze gene expression, COS-7 cells were transiently transfected with recombinant constructs using Lipofectin (25Felgner P.L. Gadek T.R. Holm M. Roman R. Chan H.W. Wenz M. Northrop J.P. Ringold G.M. Danielsen M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7413-7417Crossref PubMed Scopus (4365) Google Scholar) according to the manufacturer's instructions (Invitrogen). Briefly, the cultured COS-7 cells were seeded on 12-well tissue culture plates (1.5 × 105cells/well). The cells were allowed to attach and grow overnight in DMEM containing 5% fetal bovine serum. Cells were transfected once they reached 70% confluence. Lipofectin (0.5 μl) was incubated with plasmid DNA (∼2 μg) for 15 min in 100 μl of DMEM followed by an addition of 900 μl of DMEM. Concurrently, COS-7 cell cultures were washed with 2 ml of DMEM. The Lipofectin-DNA mixture was added to the cultures followed by incubation at 37 °C for 5 h in an incubator. The DNA-Lipofectin mixture was replaced with 1 ml of DMEM containing 5% fetal bovine serum. The cells were harvested in 400 μl of lysis buffer 3 days later, and samples of cell lysate and culture medium were frozen at −70 °C until analysis. Cell lysate and culture medium samples were prepared in loading dye and subjected to SDS-PAGE in separating gel containing 5 or 7% acrylamide. Two layer gels (12 and 5%) were used if large and small products were analyzed in the same experiment. The buffer system was 1× TG (Tris-glycine buffer according to Amresco) containing 1% SDS. Separated proteins were trans-blotted onto a nitrocellulose membrane in 1× Tris-glycine buffer containing 20% methanol at 60 V for 2 h at 4 °C. The membrane was blocked in TBST (10 mmTris-Cl, pH 8.0, 150 mm NaCl, 0.05% Tween 20) containing 5% nonfat dry milk powder (TBSTM) for 30 min at room temperature and then incubated at 4 °C overnight with the monoclonal antibody 4B6 (or biotinylated 4B6) in TBSTM. The membranes were washed with TBST (3 × 10 min washes) and then incubated for 2 h with horseradish peroxidase-conjugated goat anti-mouse IgG antibodies (1:10,000 dilution in TBSTM). After washing as above, the bound antibodies were visualized with chemiluminescence (ECL kit, Amersham Biosciences, Inc.). Trichloroacetic acid was added to culture medium or cell lysate to a final concentration of 10% followed by incubation on ice for 30 min. The precipitated proteins were pelleted by centrifugation (10,000 × g) at 4 °C for 30 min. The pelleted proteins were resuspended in loading dye and subjected to Western blot analysis as above. Protein G beads (50 μl of gel slurry) were incubated with excess 4B6 antibody at room temperature for 2 h. The unbound antibody was removed, and the gel beads were washed three times with 1 × phosphate-buffered saline. Culture media or cell lysates from COS-7 cells transfected with different constructs or control vector were mixed with 2× phosphate-buffered saline buffer in a 1:1 ratio followed by incubation with the antibody-bound gel beads at 4 °C overnight. The gel beads were extensively washed and resuspended in 200 μl of 1× phosphate-buffered saline. Chondroitinase ABC (0.2 units) was added to digest GAG chains at 37 °C for 2 h. The digested product was recovered with protein-loading dye and analyzed on Western blot probed with 4B6 as above. To study the effects of cysteines in the aggrecan G3 domain on product secretion, we tested 20 constructs in which the cysteines were subjected to mutagenesis in different combinations. There are six cysteines in the CRD motif and four in the CBP motif. Three groups of mutation constructs in CRD were generated, resulting in 12 recombinant constructs (Fig. 1). In Group I constructs, the three cysteines in the amino-terminal fragment (C1 to C3) and the three cysteines in the carboxyl-terminal fragment (C4 to C6) of CRD motif were mutated in parallel. The four resulting constructs, CRD, CRDC2C3C4C5, CRDC3C4, and CRDC0, retained, respectively, all cysteines, the middle four cysteines, the middle two cysteines, and no cysteines. In Group II, the three cysteines in the carboxyl-terminal fragment of CRD were deleted, and the three cysteines in the amino-terminal fragment were mutated, resulting in four constructs, CRDC1C2C3, CRDC1C3, CRDC2C3, and CRDC3. In Group III, the three cysteines in the amino-terminal fragment of CRD were deleted, and the three cysteines in the carboxyl-terminal fragment were mutated, producing constructs CRDC4C5C6, CRDC4C6, CRDC4C5, and CRDC4. The amino acid sequences of all constructs listed in Fig. 1 are given in Fig. 2. All amino acid sequences were deduced from the cDNA sequences after the constructs were generated. We isolated at least two clones from each expression construct to guarantee that we would obtain a sequence that was identical to the reported aggrecan sequences (11Sai S. Tanaka T. Kosher R.A. Tanzer M.L. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5081-5085Crossref PubMed Scopus (61) Google Scholar, 12Li H. Schwartz N.B. Vertel B.M. J. Biol. Chem. 1993; 268: 23504-23511Abstract Full Text PDF PubMed Google Scholar). Theunderlined amino acids (Gly or Trp) were mutated from the wild-type Cys residue. These two amino acid residues were chosen to replace Cys because of codon similarities. The 12 constructs obtained from the CRD motif were expressed in COS-7 cells. Culture medium and cell lysates were harvested and analyzed by electrophoresis and Western blot as described under “Experimental Procedures.” Probing with the monoclonal antibody 4B6 showed that all expressed constructs were of the expected sizes (Fig.3). In Group I, all constructs were well expressed and secreted except CRDC3C4, which was poorly secreted. The CRDC3C4 product was well detected in 10-fold concentrated culture medium (Fig. 3A, CRDC3C4,p). In Group II, CRDC1C2C3, CRDC1C3, CRDC2C3, and CRDC3 were well expressed, but only CRDC1C2C3 and CRDC1C3 were well secreted. The products of CRDC2C3 and CRDC3 were poorly secreted as compared with the control construct CRD. The secreted products of CRDC2C3 and CRDC3 were visible on Western blot after these samples were concentrated (10-fold) (Fig. 3B). All Group III products were well expressed and secreted to culture medium as compared with the control CRD (Fig. 3C). The G3 complement binding protein-like motif contains four cysteines. To study their effects on product secretion, we generated two more groups of constructs (Group IV and Group V, Fig. 1). In Group IV, two cysteines in the carboxyl-terminal fragment of CBP were mutated, producing CBPC1C2C4 and CBPC1C2. These constructs were well expressed and well secreted (Fig. 4A). Expression of the CBP construct or CBP containing the carboxyl-terminal tail (CBPtail) indicated that the tail is not required for product secretion (Fig. 4A). In Group V, the amino-terminal cysteine of CBP was mutated along with the two cysteines in carboxyl fragment, producing constructs CBPC2C3C4, CBPC2C4, CBPC2C3, and CBPC2. Culture medium and cell lysates from COS-7 cells transfected with these constructs were analyzed on a Western blot probed with 4B6. The product of CBPtail was used as a control. All constructs were weakly detected in cell lysates and culture medium as compared with CBPtail (Fig. 4B). After product precipitation (10-fold concentration), they were readily detected on Western blot (Fig. 4C). In addition to investigating the effects of G3-subdomain conformation on construct secretion, we aimed to dissect the functions of G3 in GAG chain attachment and product secretion. We did not observe accumulation of GAG chain-modified product in cell lysate; GAG-modified products were always secreted to culture medium, indicating the close relationship of these two processes. We have previously shown that the construct G1CSD is not modified by GAG chain and not secreted, whereas G1CSDG3 is modified by GAG chains, and the product is secreted (21Kiani C. Lee V. Cao L. Chen L. Wu Y. Zhang Y. Adams M.E. Yang B.B. Biochem. J. 2001; 354: 199-207Crossref PubMed Scopus (33) Google Scholar). We thus sought to identify element(s) in G3 that facilitate product secretion but had no effect on GAG chain attachment and elements that are not secreted on their own but enable GAG chain attachment. The current study indicated that the products of G3 subdomain constructs CRD and CBPtail were well secreted. We thus examined these fragments for GAG chain attachment by adding them to the G1CSD construct. All new G1CSD-linked constructs, now named G1CSDCRDCBP, G1CSDCRD, and G1CSDCBPtail, as well as G1CSDG3 and G1CSD controls (Fig. 5A), were well expressed (Fig. 5B). Interestingly, the G1CSDCBPtail product was not modified by GAG chains nor secreted to culture medium, although CBPtail, in the absence of G1CSD domains, is secreted on its own. On the other hand, the products of G3 and CRDCBP (Fig. 5C) and the product of CRD (Fig. 3A) were well expressed and secreted, and constructs containing these fragments (i.e. G1CSDG3, G1CSDCRDCBP, and G1CSDCRD) generated products modified by GAG chains and secreted. It was noted that the cell-associated products of the above five constructs containing the CSD fragment showed little diffusion, likely indicating low levels of GAG chain modification. The apparent molecular weights of products in cell lysate (lys) and culture medium (med) were much greater than the calculated molecular mass (cal) of the recombinant constructs (Fig. 5D). Although the apparent molecular mass of the cell lysate products was greater than the calculated molecular mass, a lack of diffusion implied that these products were not subjected to GAG addition. Rather, they may have been modified by glycosylation, as these constructs contain several potential sites for N-glycosylation (located at Asn residues 76, 122, 330, and 2082) according to the published sequence (12Li H. Schwartz N.B. Vertel B.M. J. Biol. Chem. 1993; 268: 23504-23511Abstract Full Text PDF PubMed Google Scholar). Thus, all constructs containing the CBP motif have four potential sites for glycosylation, whereas constructs containing the G1 domain have three potential sites for glycosylation. Because glycosylation of these recombinant constructs appeared not to be affected by the presence of different G3 subdomains, no further study was conducted. In contrast to the cell lysate products, the products in the culture medium migrated diffusely on Western blot, resulting in broad ranges of molecular mass. This may have been caused by GAG chain attachment to core protein, as the core proteins of the recombinant constructs contains eight potential sites for GAG chain modification. To confirm the presence of GAG chains, we treated these products with chondroitinase ABC. Treated products migrated as single bands on Western blot, confirming this explanation (Fig. 5E). The addition of GAG chains appeared to be affected by the addition of different G3 subdomains. The role of the cysteines in the CRD and CBP motifs was further studied by adding the G1CSD fragment to mutant constructs CRDC0, CRDC3, CRDC4, CBPC2, and CBPC1C2 used in the experiments described above. These constructs were labeled G1CSDCRDC0, G1CSDCRDC3, G1CSDCRDC4, G1CSDCBPC2, and G1CSDCBPC1C2 (Fig. 6A). Before the addition of G1CSD, the mutant CRDC3 was notably ill-secreted (Fig. 3); with the addition of CRDC3 to G1CSD, the new construct (G1CSDCRDC3) was well expressed, and the products were modified by GAG chains (as indicated by diffusion of the secreted products) and secreted to culture medium (Fig. 6B). The cell-associated products showed little diffusion of bands (little GAG chain modification). Again, the apparent molecular masses of the products in cell lysate (lys) and culture medium (med) were much greater than the calculated molecular mass (cal) of each recombinant construct (Fig. 6C). Chondroitinase ABC treatment further confirmed that the secreted recombinant proteoglycans were subjected to post-translational modification by GAG chains as expected (Fig. 6D). Taken together, we have studied five types of G1CSD-containing constructs differing in GAG chain modification and product secretion (Figs. 5 and 6). All results are summarized in TableII. (i) Without any components from the G3 domain, the product of G1CSD was not modified by GAG chains nor secreted. (ii) The addition of certain elements from the G3 domain to the G1CSD construct, including G3, CRD, CRDCBP, CRDC0, CRDC4, and CBPC1C2, allowed GAG chain modification and product secretion. All of these G3 elements, when linked to the leading peptide, were well expressed and secreted. (iii) The construct CRDC3 was well expressed but poorly secreted, but the addition of the CRDC3 fragment to G1CSD allowed GAG modification and product secretion. (iv) The construct CBPC2 is poorly expressed and poorly secreted, but the addition of CBPC2 to G1CSD allowed GAG modification and" @default.
• W2002167434 created "2016-06-24" @default.
• W2002167434 creator A5012710443 @default.
• W2002167434 creator A5014431860 @default.
• W2002167434 creator A5028932344 @default.
• W2002167434 creator A5058008919 @default.
• W2002167434 creator A5063942792 @default.
• W2002167434 creator A5064715130 @default.
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• W2002167434 date "2002-01-01" @default.
• W2002167434 modified "2023-09-29" @default.
• W2002167434 title "The Folded Modules of Aggrecan G3 Domain Exert Two Separable Functions in Glycosaminoglycan Modification and Product Secretion" @default.
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