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• W2010706444 abstract "Aggrecan, a major structural proteoglycan in cartilage, contains three globular domains, G1, G2, and G3, as well as sequences for glycosaminoglycan modification. A large number of proteases are implicated in aggrecan cleavage in normal metabolism, aging, and arthritis. These proteases are known to cleave at the IGD, KS, and CS domains. Here we report for the first time evidence of cleavage at a novel site, the carboxyl tail of aggrecan. Results from deletion mutants of the tail indicated that the likely cleavage sites were two consensus sequences, RRLXK and RSPR, present in the aggrecan analogs of many species. This was confirmed by site-directed mutagenesis. A construct containing two G3 domains (G3G3) was also found to cleave between the G3 duplicates. When G3 tail was linked to a glycosaminoglycan-modifying sequence, it was protected from cleavage. Furin inhibitor also reduced the levels of tail cleavage. The carboxyl tails of chicken and human versican were not cleaved, despite the presence of the consensus sequence. Our studies indicate that the basic amino acids present in the tail play an important role in cleavage, and this mechanism is specific to aggrecan. Aggrecan, a major structural proteoglycan in cartilage, contains three globular domains, G1, G2, and G3, as well as sequences for glycosaminoglycan modification. A large number of proteases are implicated in aggrecan cleavage in normal metabolism, aging, and arthritis. These proteases are known to cleave at the IGD, KS, and CS domains. Here we report for the first time evidence of cleavage at a novel site, the carboxyl tail of aggrecan. Results from deletion mutants of the tail indicated that the likely cleavage sites were two consensus sequences, RRLXK and RSPR, present in the aggrecan analogs of many species. This was confirmed by site-directed mutagenesis. A construct containing two G3 domains (G3G3) was also found to cleave between the G3 duplicates. When G3 tail was linked to a glycosaminoglycan-modifying sequence, it was protected from cleavage. Furin inhibitor also reduced the levels of tail cleavage. The carboxyl tails of chicken and human versican were not cleaved, despite the presence of the consensus sequence. Our studies indicate that the basic amino acids present in the tail play an important role in cleavage, and this mechanism is specific to aggrecan. chondroitin sulfate-modifying sequence keratan sulfate-modifying sequence carbohydrate recognition domain complement binding protein glycosaminoglycan Dulbecco's modified Eagle's medium fetal bovine serum phosphate-buffered saline leading peptide matrix metalloproteinase horseradish peroxidase Members of the family of large aggregating chondroitin sulfate proteoglycans include aggrecan, versican, neurocan, and brevican. All are composed of a signal peptide, a globular domain named G1, a large fragment for chondroitin sulfate modification (CS1 sequence), and a globular domain G3 (1Margolis R.U. Margolis R.K. Methods Enzymol. 1994; 245: 105-126Crossref PubMed Scopus (89) Google Scholar). The G1 domain, which is homologous to link protein, contains one immunoglobulin (IgG)-like domain and two tandem repeat motifs (2Li H. Schwartz N.B. Vertel B.M. J. Biol. Chem. 1993; 268: 23504-23511Abstract Full Text PDF PubMed Google Scholar, 3Neame P.J. Barry F.P. Experientia. 1993; 49: 393-402Crossref PubMed Scopus (110) Google Scholar, 4Perkins S.J. Nealis A.S. Dudhia J. Hardingham T.E. J. Mol. Biol. 1989; 206: 737-748Crossref PubMed Scopus (71) Google Scholar). Aggrecan is unique in containing an additional globular domain G2 that is structurally similar to the tandem repeats of the G1 domain. An inter-globular domain (IGD) is located between the G1 and G2 domains, and a segment modified by keratan sulfate (KS sequence) is situated between the G2 domain and the CS sequence. The G3 domain is composed of one or two alternatively spliced epidermal growth factor (EGF)-like motif(s), one lectin (also called carbohydrate recognition domain or CRD)-like motif, one complement binding protein (CBP)-like motif, and a short C-terminal tail (2Li H. Schwartz N.B. Vertel B.M. J. Biol. Chem. 1993; 268: 23504-23511Abstract Full Text PDF PubMed Google Scholar, 5Chandrasekaran L. Tanzer M.L. Biochem. J. 1992; 288: 903-910Crossref PubMed Scopus (30) Google Scholar, 6Lohmander L.S. Neame P.J. Sandy J.D. Arthritis Rheum. 1993; 6: 1214-1222Crossref Scopus (382) Google Scholar). The structures of these motifs, especially the tail, are highly conserved in aggrecan across species boundaries. In the early stages of cartilage development, versican is the predominant proteoglycan and is believed to play an important role in tissue development. After cartilage maturation, versican is replaced by aggrecan, which facilitates the formation of a matrix network for resilience and load-bearing. Aggrecan processing seems to be regulated, in part, by the G3 domain. This was initially observed in the chicken disorder nanomelia, which produces a lethal phenotype in homozygous form (due to failure of chondrogenesis and osteogenesis) and dwarfism in heterozygote form (7Vertel 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). Cartilage of the nanomelic mutant lacks aggrecan in its matrix. It was discovered that the core protein of this mutant aggrecan is truncated as a result of a premature stop codon at the N-terminal side of G3 (2Li H. Schwartz N.B. Vertel B.M. J. Biol. Chem. 1993; 268: 23504-23511Abstract Full Text PDF PubMed Google Scholar). In addition, its secretion is hindered and no modification by glycosaminoglycan (GAG) chains occurs (7Vertel 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). Recently, it was reported that the G3 and G1 domains play roles in processing of recombinant aggrecan and versican (8Day 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, 9Domowicz M.S. Pirok 3rd, 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, 10Kiani 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, 11Luo 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, 12Yang 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, 13Zheng J. Luo W. Tanzer M.L. J. Biol. Chem. 1998; 273: 12999-13006Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 14Chen L., Wu, Y. Lee V. Kiani C. Yao Y. Adams M.E. Yang B.B. J. Biol. Chem. 2002; 277: 2657-2665Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Extensive aggrecan degradation occurs during normal cartilage metabolism, aging, and joint diseases (15Fosang A.J. Neame P.J. Hardingham T.E. Murphy G. Hamilton J.A. J. Biol. Chem. 1991; 266: 15579-15582Abstract Full Text PDF PubMed Google Scholar, 16Ilic M.Z. Handley C.J. Robinson H.C. Mok M.T. Arch. Biochem. Biophys. 1992; 294: 115-122Crossref PubMed Scopus (164) Google Scholar). In mature cartilage, up to half of the aggrecan population loses its G3 domain. The other domains are also subjected to cleavage, resulting in the release of degraded fragments in the matrix. The exception is the G1 domain, which is trapped and retained by hyaluronan. Loss of aggrecan is a major feature of cartilage degradation associated with arthritis (6Lohmander L.S. Neame P.J. Sandy J.D. Arthritis Rheum. 1993; 6: 1214-1222Crossref Scopus (382) Google Scholar). In the early stages of rheumatoid arthritis, the chondroitin sulfate-bearing sequence of aggrecan, the CS domain, is preferentially released. The synovial fluid of patients with osteoarthritis and joint injury contains fragments of degraded aggrecan (17Lohmander L.S. J. Rheumatol. 1995; 43: 75-77Google Scholar). A large number of proteases are known to cleave aggrecan, and their target sites are located in the IGD, KS, and CS domains (15Fosang A.J. Neame P.J. Hardingham T.E. Murphy G. Hamilton J.A. J. Biol. Chem. 1991; 266: 15579-15582Abstract Full Text PDF PubMed Google Scholar, 18Flannery C.R. Lark M.W. Sandy J.D. J. Biol. Chem. 1992; 267: 1008-1014Abstract Full Text PDF PubMed Google Scholar, 19Lark M.W. Gordy J.T. Weidner J.R. Ayala J. Kimura J.H. Williams H.R. Mumford R.A. Flannery C.R Carlson S.S. Iwata M. J. Biol. Chem. 1995; 270: 2550-2556Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 20Sandy J.D. Flannery C.R. Neame P.J. Lohmander L.S. J. Clin. Invest. 1992; 89: 1512-1516Crossref PubMed Scopus (388) Google Scholar, 21Singer I.I. Kawka D.W. Bayne E.K. Donatelli S.A. Weidner J.R. Williams H.R. Ayala J.M. Mumford R.A. Lark M.W. Glant T.T. Nabozny G.H. David C.S. J. Clin. Invest. 1995; 95: 2178-2186Crossref PubMed Scopus (114) Google Scholar). No protease has yet been reported to cleave inside the globular domains, or in the carboxyl tail. Our preliminary studies suggested that the carboxyl tail of aggrecan is removed during product secretion. After transfecting COS-7 cells with a His-tagged (at the C terminus) G3 construct, we did not detect the His-tagged G3 product in culture medium but detected this product in cell lysate. An antibody against another epitope at the amino region allowed detection of product in both culture medium and cell lysate. Hence, we hypothesized that the G3 tail was removed after product secretion. The present study was designed to determine the mechanism of this process, to identify amino acid motifs that may be involved in this degradation, and to determine whether this process is specific to aggrecan or common to the proteoglycan family. PCR amplification kit, Taq DNA polymerase, and restriction endonucleases were from Roche Molecular Biochemicals and New England BioLabs. DNA marker was from Fermentas MBI. Bacterial growth medium was from Difco. Prestained protein marker was from New England BioLabs. Lipofectin, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), Hanks' balanced salt solution, trypsin/EDTA, and T4 DNA ligase were from Invitrogen. ECL Western blot detection kit was from Amersham Biosciences, Inc. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was from Sigma Chemical Co. Anti-His-tag monoclonal antibody and DNA Midi-prep kit were purchased from Qiagen Inc. Tissue culture plates (6-well, 12-well, and 100 mm) were from Nunc Inc. Protease inhibitors Amastatin and E64 were from Calbiochem. A furin inhibitor (decanoyl-RVKR-chloromethylketone) was from Bachem (King of Prussia, PA). All other chemicals were from Sigma. COS-7 cells were from American Type Culture Collection. The cells were cultured in DMEM supplemented with 5% FBS at 37 °C in a humidified incubator containing 5% CO2. In this study, a total of 43 recombinant constructs was used: G3His, HisG3, C2 (C representing the last cysteine residue of aggrecan), C7, C13, C16, C20, C2tail, C7tail, C13tail, C16tail, C20tail, Muttail, CR8P, CR9P, CK12A, CR13S, del8–12, delR13S, delR16P, G3KRTS, G3allmut, HuC8–13, BoC8–13, HuC13–16, HuC16–19, G3KS, G3G3, C2G3, C7G3, C13G3, CR9PG3, CK12AG3, CR13SG3, G3KRTSG3, G3allmutG3, del8–12G3, huC8–13G3, cvG3aG3, hvG3aG3, cvRSPRaG3, hvRSPRaG3, and cvG3ΔEGFHis. G3His, G3KS, and cvG3ΔEGFHis have been described in our previous publications (10Kiani 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, 22Yang B.B. Zhang Y. Cao L. Yang B.L. Matrix Biol. 1997; 16: 541-561Crossref Google Scholar, 23Zhang Y. Cao L. Yang B.L. Yang B.B. J. Biol. Chem. 1998; 273: 21342-21352Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 24Yang B.L. Zhang Y. Cao L. Yang B.B. J. Cell. Biochem. 1999; 72: 210-220Crossref PubMed Scopus (92) Google Scholar, 25Zhang 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; for structures see Figs. 1, 6, and 9 of this paper). The G3His construct was generated by linking together the leading peptide (LP) of link protein, aggrecan G3 domain and a His-tag. The leading peptide contained link protein signal peptide and an epitope recognized by the monoclonal antibody 4B6 (26Binette F. Cravens J. Kahoussi B. Haudenschild D.R. Goetinck P.F. J. Biol. Chem. 1994; 269: 19116-19122Abstract Full Text PDF PubMed Google Scholar). The G3KS construct contained the leading peptide, aggrecan G3 domain, aggrecan KS domain, and a His-tag.Figure 6Cleavage of G3 tail inhibited by glycosaminoglycan chains. Cell lysate and culture medium from G3G3 and G3KS were analyzed on Western blot probed with 4B6. The G3G3 products in the lysate had the expected size. However, most of the secreted G3G3 product had a size similar to that of G3His. The faint band at 70 kDa probably represents intact G3G3. The G3KS product also exhibited the expected size, and the smear (the larger sizes) suggested GAG chain attachment to the KS sequence. No cleavage was observed for G3KS product.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 9Human and chicken versican contain the consensus sequence RXXR in their G3 tail but are not cleaved. A, linking native human or chicken versican G3 cDNA with the aggrecan G3 domain produced hvG3aG3 and cvG3aG3 constructs. Two additional constructs (hvRSPRaG3 and cvRSPRaG3) were engineered to contain the consensus sequence RSPR in the tails of human and chicken versican G3 domains. B, cell lysate (L) and culture medium (M) of hvG3aG3, cvG3aG3, cvRSPRaG3, hvRSPRaG3, and G3G3 were analyzed on Western blot probed with 4B6. Little product cleavage was detected in the culture medium from hvG3aG3-, cvG3aG3-, hvRSPRaG3-, and cvRSPRaG3-transfected cells, but the majority (∼90%) of aggrecan G3G3 product was cleaved.C, bovine chondrocytes were transiently transfected with G3G3, G3allmutG3, cvG3aG3, hvG3aG3, and a control vector pcDNA3. Cell lysate (L) and culture medium (M) were analyzed on Western blot probed with 4B6. All constructs were well expressed and secreted. The G3G3 product in the culture medium was completely cleaved, and a small amount of the cell-associated G3G3 product was cleaved. D, the constructs G3His and cvG3ΔEGFHis (from chicken versican G3 domain) were expressed in COS-7 cells. Cell lysate (L) and culture medium (M) were analyzed on Western blot probed with anti-His antibody. Only the His epitope in the secreted product of cvG3ΔEGFHis (but not of G3His) was detected.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The HisG3 construct was generated by ligating two fragments, the leading peptide containing a His-tag and the G3 domain, into pcDNA3. The leading peptide containing a His-tag was produced using two primers, LP40CHisXhoI and LPNKozakEcoRI (for sequences see Table I), in a PCR using link protein as a template. The PCR product was doubly digested withEcoRI and XhoI, purified, and ligated with the G3 fragment, which was derived by digesting the G3His construct, into pcDNA3.Table ISequence and restriction endonuclease sites for oligonucleotides Open table in a new tab The remaining 39 constructs were based on the G3His construct. They all contain the leading peptide, the CRD and CBP motifs, a modified tail, and the His-tag. Modification of the tail was performed through either systematic incremental deletion, site-directed mutagenesis, or engineering of a potential site for cleavage from other species or proteoglycans into the tail. Ten constructs were made to contain incremental deletion of the tail. Two types of deletion were made. In the first type, the entire tail was incrementally deleted producing 5 constructs (C2, C7, C13, C16, and C20). This type was based on the plasmid Type I (see Fig.2 A). The end of the tail contains a restriction enzyme siteXbaI (encoding two amino acids SR) followed by a His-tag. The original XbaI site on the vector was inactivated by ligation with the restriction enzyme site NheI. The primersNheIHisXbaIXhoI and CRDNXhoI were used in a PCR with aggrecan G3 domain as a template. The product was purified, digested with XhoI andNheI, and inserted into XhoI- andXbaI-digested G3His construct to generate the Type I construct. To generate incremental deletion mutations, two primers were used in each PCR: one complementary to the 5′ terminus of G3 domain (CRDNXhoI) whereas the other was complementary to the desired 3′ region of the tail. For example, primer C2CXbaI and CRDNXhoI were used in a PCR using G3 as template. The PCR product was purified, doubly digested with XhoI andXbaI, and ligated with XhoI- andXbaI-digested Type I plasmid to produce construct C2. Primer C7CXbaI was combined with CRDNXhoI to produce the construct C7. C13CXbaI was used for production of construct C13, whereas C16CXbaI was used for construct C16 and C20CXbaI was used for construct C20 (see Fig.2 B). To produce the second type of incremental deletion, nine amino acids from the extreme 3′-end of the tail were linked to the His-tag in a PCR, using the primers XbaItailHis and pcDNA3XmaI (complementary to nucleotides 2091–2096 of the vector). The PCR product was digested with XbaI andXmaI and inserted into XbaI- andXmaI-digested construct C2 resulting in the construct C2tail. Replacement of the XbaI-XmaI fragment from the construct C7 with the PCR product produced construct C7tail. Constructs C13tail, C16tail, and C20tail were generated in the same way. Another ten constructs contained site-directed mutagenesis in the tail that could be divided into three groups. In Group 1, the first potential consensus sequence (amino acids RRLYKR, see Fig.3 A) was mutated: in each construct, one basic amino acid was mutated to a non-basic amino acid. For example, two primers, CSDFBamHI and CR8PXbaI, were used in a PCR with C13tail as a template. The primer CR8PXbaI generated a single mutation at amino acid C8 (R → P). The PCR product was purified, doubly digested with XhoI and XbaI, and ligated into XhoI- and XbaI-digested vector from the construct C13tail. Using primers CR9PXbaI and CSDFBamHI, we generated a single mutation at amino acid C9 (R → P) and produced the construct CR9P. Likewise, combined with CSDFBamHI, primers CK12AXbaI and CR13SXbaI were used to generate constructs CK12A and CR13S, respectively. In the second group, we linked the second consensus sequence to construct C2tail (see Fig. 3 A). This was performed by using two primers, CSDFBamHI and del8–12, which deleted the first motif, amino acids 8–12, in a PCR with G3 domain as a template. The PCR product was purified, digested withXhoI and XbaI, and ligated into XhoI- and XbaI-digested C2tail. Combined with primer CSDFBamHI, primer delR13SXbaI was used to generate a mutation at amino acid 13 to produce the construct delR13S, and primer delR16PXbaI was used to generate a mutation at amino acid 16 to produce the construct delR16P. In the third group, each construct contained multiple mutations. Construct Muttail was generated in a PCR using two primers, CSDFBamHI and muttailXbaI. The PCR product was inserted intoXhoI- and XbaI-digested construct C20. Construct G3KRTS was generated using two primers CSDFBamHI and G3KRTSXbaI, and, after similar treatment, the PCR product was inserted into XhoI- and XbaI-digested plasmid C20tail. Using a similar method, the primers G3allmutXbaI and CSDFBamHI were used to produce the construct G3allmut (see Fig. 4 A).Figure 4Mutation of all basic amino acid residues completely inhibits tail cleavage. A, site-directed mutagenesis was carried out at multiple sites to remove important basic amino acids in the G3 tail. The first construct (G3KRTS) contained mutations in C12 (K → T) and C13 (R → S). The second construct (G3allmut) contained mutations of 6 amino acids (boldface).B, cell lysate (L) and culture medium (M) from cultures transfected with these two constructs and C2tail (as control) were analyzed on Western blot. 4B6 probing showed that all constructs were well expressed. Probing with anti-His antibody revealed consistent levels of products in cell lysate. However, the amount of secreted products from G3KRTS-transfected was only 10% of that from C2tail-transfected cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Four constructs contained consensus sequences obtained from bovine, human, dog, mouse, or rat aggrecan (see Fig. 5). These constructs have similar structure: the putative cleavage motif was engineered into the construct C7tail. For example, the sequence RRLQKR from the first potential cleavage site of rat, human, or dog was linked to 3′ of the construct C2 by using two primers, CSDFBamHI and HuC8–13XbaI, in a PCR. After treatment similar that described above, the PCR product was inserted into XhoI- andXbaI-digested plasmid C7tail to produce the chimeric construct HuC8–13. The sequence RHLQKR (from bovine potential cleavage site) was linked to the 3′ of the construct C7 using the primers BoC8–13XbaI and CSDFBamHI to generate the chimeric construct BoC8–13. The second potential protease site, RSSR, from rat, human, or dog was linked to 3′ of the construct C7 using primers HuC13–16XbaI and CSDFBamHI to produce the chimeric construct HuC13–16. The third potential protease site in human aggrecan, RHPR, was linked to 3′ of the construct C7 using primer HuC16–19XbaI and CSDFBamHI to generate the chimeric construct HuC16–19. Fifteen constructs are based on a G3-G3 duplex. The G3G3 construct was generated by replacing the His-tag in the G3His construct with a G3His fragment (see Fig. 6). Two separate aliquots of the G3His construct were doubly digested with (i) EcoRI and SalI and (ii) XhoI and EcoRI to purify LP60G3 and G3His+pcDNA3, respectively. The two fragments were then ligated together. Ten constructs (C2G3, C7G3, C13G3, CR9PG3, CK12AG3, CR13SG3, G3KRTSG3, G3allmutG3, del8–12G3, huC8–13G3) contained the mutated or truncated G3 in addition to normal aggrecan G3 (see Fig. 8). This was achieved by replacing the junction of the mutated or truncated G3 and pcDNA3 vector (XbaI-ApaI) withXbaI-ApaI-containing normal G3. The latter G3 was obtained in a PCR using aG3NXbaI and aG3CApaI as primers. The cvG3aG3 and hvG3aG3 were produced by linking the chicken versican G3 domain or human versican G3 domain with the normal aggrecan G3 domain as above (see Fig. 9). Generation of chicken versican G3 domain has been described by us previously (27Zhang 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, 28Zhang Y., Wu, Y. Cao L. Lee V. Chen L. Lin Z. Kiani C. Adams M.E. Yang B.B. Exp. Cell Res. 2001; 263: 33-42Crossref PubMed Scopus (41) Google Scholar). We used an reverse transcriptase-PCR (with hvG3NXhoI and hvG3CXbaI as primers and glioma total RNA as template) to produce human versican G3 domain. Finally, we engineered the consensus sequence RSPR in the tail of chicken and human versican G3 domains (in constructs cvG3aG3 and hvG3aG3, respectively) using primers cvG3CRSPRXbaI and hvG3CRSPRXbaI in PCR reactions. DNA was amplified in PCRs 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. The reactions were carried out at 94 °C for 5 min for one 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 then doubly digested with two appropriate restriction endonucleases. The DNA was ligated into the linearized plasmid pcDNA3. The ligation reaction was carried out at 16 °C overnight. The ligation mixture was used for transformation of competent Escherichia colistrain DH5α. After clone selection, 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 sequence. To analyze gene expression, COS-7 cells or bovine chondrocytes were transfected transiently with recombinant constructs using Lipofectin according to the manufacturer's instructions (Invitrogen). Briefly, the cultured COS-7 cells or bovine chondrocytes were seeded onto 12-well tissue culture plates (1.5 × 105 cells/well). The cells were allowed to attach and grow overnight in DMEM containing 5% FBS. Cells were transfected once they reached 70% confluence. Lipofectin (2 μl) was incubated with plasmid DNA (∼2 μg) for 15 min in 100 μl of DMEM followed by the addition of 900 μl of DMEM. Concurrently, COS-7 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% FBS. Three days later, culture medium was harvested, and dead and floating cells were removed by centrifugation. Cell lysate in each well was obtained by using 200 μl of lysis buffer. Because the volume of medium in each well is 5-fold the volume of lysis buffer used, the percentage of the portions of cell lysate used in each loading well (see below) represented 5-fold more than that of the medium. Cell lysate and culture medium were subjected to SDS-PAGE in separating gel containing 10–12% acrylamide. The buffer system was 1× TG (Tris-glycine buffer according to Amresco) containing 1% SDS. Separated proteins were transblotted onto a nitrocellulose membrane in 1× TG buffer containing 20% methanol at 60 V for 2 h in a cold room. The membrane was blocked in TBST (10 mm Tris-Cl, pH 8. 0, 150 mm NaCl, 0.05% Tween 20) containing 10% nonfat dry milk powder (TBSTM) for 30 min at room temperature, and then incubated at 4 °C overnight with the monoclonal antibody 4B6 or the anti-His-tag monoclonal antibody in TBSTM. The membranes were washed with TBST (3 × 30-min washes) and then incubated for 2 h with HRP-conjugated goat anti-mouse IgG antibody (1:50,000 dilution) in TBSTM. After washing as above, the bound antibody were visualized with chemiluminescence (ECL kit, Amersham Biosciences, Inc.). To compare the G3 tail cleavage, it was necessary to load an equal amount of secreted products to each well on Western blot. To do so, the products of each construct, as well as one control (product of C2tail construct) were pre-analyzed on Western blot probed with 4B6, which is much more sensitive than the anti-His antibody. Equal amounts of gene products, obtained by dilution, were then analyzed for G3 tail cleavage probed with anti-His antibody. Because the affinity of anti-His antibody is much lower than the 4B6 antibody, we had to use larger amounts of products (∼ 3-fold) for anti-His staining. A reduction in the intensity of bands probed with anti-His antibody would suggest G3 tail cleavage. COS-7 cells were transiently transfected with G3G3 construct. After overnight incubation, culture medium was replaced with fresh medium containing 5% FBS and furin inhibitor decanoyl-RVKR-chloromethylketone. Protease inhibitors Amastatin and E64 were used as controls. Cell lysate and culture medium were harvested and analyzed on Western blot as above. Relative protein concentration was estimated by using a densitometer (Molecular Dynamics) according to the manufacturer's instructions for scanning the densities of the signals on the films after Western blot. The relative intensity of each band after Western blot development is shown below the blot. In the case of product cleavage in duplex constructs, both protein bands (undegraded and degraded) were scanned and density was calculated using the following formula: degradation (%) = OD of degraded band/(OD of degraded band + OD of undegraded band). To assess the possibility of cleavage of the C-terminal tail of the chicken aggrecan G3 domain, we initially generated a G3 construct that contains the leading peptide of link protein at the N terminus. This leading peptide harbors the signal peptide and an epitope recognized by the monoclonal antibody 4B6 (26Binette F. Cravens J. Kahoussi B. Haudenschild D.R. Goetinck P.F. J. Biol. Chem. 1994; 269: 19116-19122Abstract Full Text PDF PubMed Google Scholar). At the C-terminal end of G3, a His-tag recognized by anti-His monoclonal antibody was added (G3His, Fig.1 A). After transfection of COS-7 cells with this G3 construct, the 4B6 antibody detected products (∼48 kDa) in both culture medium and cell lysate on Western blot (Fig. 1 B). However, the anti-His antibody failed to detect the product in culture medium, but could detect the product (∼48 kDa) in cell lysate (Fig. 1 C). This suggested that the G3 tail has been removed after product secretion. To test this, we generated another construct (HisG3) containing both the 4B6 epitope and the His-tag at the N-terminal region of the construct. Analysis of cell lysate and culture media from COS-7 cells transfected with HisG3 on Western blot indicated that products of HisG3 (∼48 kDa) were detec" @default.
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• W2010706444 title "Cleavage of the Carboxyl Tail from the G3 Domain of Aggrecan but Not Versican and Identification of the Amino Acids Involved in the Degradation" @default.
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• W2010706444 doi "https://doi.org/10.1074/jbc.m110227200" @default.
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