Link Protein Has Greater Affinity for Versican than Aggrecan

Abstract

The function of link protein in stabilizing the interaction between aggrecan and hyaluronan to form aggrecan aggregates, via the binding of link protein to the aggrecan G1 domain and hyaluronan, is well established. However, it is not known whether link protein can function with similar avidity with versican, another member of the large hyaluronan-binding proteoglycan family that also binds to hyaluronan via its G1 domain. To address this issue, we have compared the interaction of the versican and aggrecan G1 domains with link protein and hyaluronan using recombinant proteins expressed in insect cells and BIAcore analysis. The results showed that link protein could significantly improve the binding of both G1 domains to hyaluronan and that its interaction with VG1 is of a higher affinity than that with AG1. These observations suggest that link protein may function as a stabilizer of the interaction, not only between aggrecan and hyaluronan in cartilage, but also between versican and hyaluronan in many tissues. The function of link protein in stabilizing the interaction between aggrecan and hyaluronan to form aggrecan aggregates, via the binding of link protein to the aggrecan G1 domain and hyaluronan, is well established. However, it is not known whether link protein can function with similar avidity with versican, another member of the large hyaluronan-binding proteoglycan family that also binds to hyaluronan via its G1 domain. To address this issue, we have compared the interaction of the versican and aggrecan G1 domains with link protein and hyaluronan using recombinant proteins expressed in insect cells and BIAcore analysis. The results showed that link protein could significantly improve the binding of both G1 domains to hyaluronan and that its interaction with VG1 is of a higher affinity than that with AG1. These observations suggest that link protein may function as a stabilizer of the interaction, not only between aggrecan and hyaluronan in cartilage, but also between versican and hyaluronan in many tissues. The family of large aggregating proteoglycans (hyalectans) includes aggrecan, versican, neurocan, and brevican (1Iozzo R.V. Annu. Rev. Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1335) Google Scholar). Although aggrecan is most abundant in cartilage, neurocan and brevican are largely restricted to nervous tissue, and versican has a rather wide tissue distribution. Versican has been identified in loose connective tissues, in fibrous, articular, and elastic cartilages, in the central and peripheral nervous system, in the epidermis, and in all three wall layers of veins and elastic arteries (2Bode-Lesniewska B. Dours-Zimmermann M.T. Odermatt B.F. Briner J. Heitz P.U. Zimmermann D.R. J. Histochem. Cytochem. 1996; 44: 303-312Crossref PubMed Scopus (162) Google Scholar). These large aggregating proteoglycans have common structural features. Their core proteins are composed of terminal globular (G) 1The abbreviations used are: G, globular; AG1, aggrecan G1 globular domain; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; ELISA, enzyme-linked immunosorbent assay; HA, hyaluronan; LP, link protein; PBS, phosphate-buffered saline; RU, response unit; SPR, surface plasmon resonance; VG1, versican G1 globular domain. 1The abbreviations used are: G, globular; AG1, aggrecan G1 globular domain; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; ELISA, enzyme-linked immunosorbent assay; HA, hyaluronan; LP, link protein; PBS, phosphate-buffered saline; RU, response unit; SPR, surface plasmon resonance; VG1, versican G1 globular domain. domains and a central chondroitin sulfate attachment domain. The N-terminal G1 domain contains an Ig-like motif, which mediates protein-protein interaction, and two link protein-type modules that are responsible for binding to hyaluronan (HA). The C-terminal G3 domain consists of one or two EGF repeats, a C-type lectin-like motif, and a complement regulatory protein-like repeat (3Yamaguchi Y. Cell Mol. Life Sci. 2000; 57: 276-289Crossref PubMed Scopus (514) Google Scholar). Link protein (LP) is a major structural component of cartilage and, like versican, is also present in many other tissues (4Neame P.J. Barry F.P. Experimentia (Basel). 1993; 49: 393-402Crossref PubMed Scopus (110) Google Scholar). Its amino acid sequence exhibits considerable homology with the G1 domains of the large aggregating proteoglycans, including conservation of five disulfide bonds. LP binds to the G1 domain of aggrecan and to HA, and its major function is thought to be the stabilization of the interaction between aggrecan and HA in the aggrecan·HA·LP ternary complex in cartilage (5Hardingham T.E. Biochem. J. 1979; 177: 237-247Crossref PubMed Scopus (232) Google Scholar). However, the function of LP in non-cartilaginous tissues devoid of aggrecan is less clear. The high degree of homology shared by the G1 domains of the different aggregating proteoglycans suggests that LP may also bind to their G1 domains and to HA to produce stable proteoglycan aggregates. In this study, we compare the interaction affinities of versican G1 (VG1), aggrecan G1 (AG1) and LP with each other and with HA using recombinant proteins expressed in insect cells via a baculovirus expression system. Our results demonstrate that LP has a higher affinity for VG1 than for AG1. We also demonstrate that LP stabilizes versican aggregates in addition to stabilizing aggrecan aggregates, its traditionally accepted role. Construction of DNA Fragments and Recombinant Donor Plasmids—A DNA fragment encoding residues 1–343 (LHK.. .to... DSE) of mature human versican (V1 splice variant of the versican gene (6Shinomura T. Nishida Y. Ito K. Kimata K. J. Biol. Chem. 1993; 268: 14461-14469Abstract Full Text PDF PubMed Google Scholar)) plus its signal peptide (7Zimmermann D.R. Ruoslahti E. EMBO J. 1989; 8: 2975-2981Crossref PubMed Scopus (500) Google Scholar) and a C-terminal His6 tag was generated from a human keratinocyte RNA preparation by reverse transcription and PCR using the primers VF and VR (Table I) as described previously (8Shi S. Ciurli C. Cartman A. Pidoux I. Poole A.R. Zhang Y. Arthritis Rheum. 2003; 48: 2903-2915Crossref PubMed Scopus (45) Google Scholar). A DNA fragment encoding residues 1–333 (VET... to... TGE) of mature human aggrecan plus its signal peptide (9Doege K.J. Sasaki M. Kimura T. Yamada Y. J. Biol. Chem. 1991; 266: 894-902Abstract Full Text PDF PubMed Google Scholar) and a C-terminal His6 tag was generated from a human chondrocyte RNA preparation by reverse transcription-PCR using the primers AF and AR. Similarly, a DNA fragment encoding human LP plus its signal peptide (10Dudhia J. Hardingham T.E. Nucleic Acids Res. 1990; 18: 2214Crossref PubMed Scopus (10) Google Scholar) and a C-terminal His6 tag was also generated from the human chondrocyte RNA preparation by reverse transcription-PCR using the primers LPF and LPR. Human primary culture keratinocytes were purchased from Colnetics Co. (San Diego). Human chondrocytes were isolated from human articular cartilage obtained at autopsy. Total RNA was extracted using TriZol® reagent (Invitrogen) according to the manufacturer's instructions. First strand cDNA was synthesized using the SuperScript first strand synthesis system (Invitrogen) and used as a template for PCR. The amplified PCR products were cloned into the pCR2.1 vector using a TA cloning kit (Invitrogen), and their authenticity was confirmed by DNA sequencing. To express recombinant VG1, AG1, and LP in insect cells using the Bac-to-Bac Baculovirus Expression System (Invitrogen), the DNA fragments were subcloned into the pFastBac1 donor vector between the BamHI and EcoRI sites to obtain the recombinant donor plasmids pFastBac-VG1, pFastBac-AG1, and pFastBac-LP. Construction of a DNA fragment encoding for honeybee melittin signal peptide (11Tessier D.C. Thomas D.Y. Khouri H.E. Laliberte F. Vernet T. Gene (Amst.). 1991; 98: 177-183Crossref PubMed Scopus (248) Google Scholar), mature LP, and a C-terminal His tag, was accomplished in two steps. First, a DNA fragment encoding for mature LP and a C-terminal His tag was PCR amplified from pFastBac-LP using primers LPFa and LPR and cloned into a TA vector. This fragment was then subcloned into pMelBac (B) (Invitrogen) between the BamHI and EcoRI sites to obtain pMelBac (B)-LP. Second, the DNA fragment encoding honeybee melittin signal peptide, mature LP, and a C-terminal His tag was PCR amplified from pMelBac (B)-LP using primers HMSPF and LPR. The resulting PCR product was also cloned into a TA vector and then subcloned into pFastBac vector at the EcoRI site.Table IPrimers used to prepare human VG1, AG1, and LP constructsPrimerSequenceVF5′-GGATCCAAGCCGCCTTCCAAGGCCAAGATGTTC-3′VR5′-GAATTCTCAATGGTGATGGTGATGATGTTCTGAATCTATTGGATGACCAATTAC-3′AF5′-GGATCCACTATGGCCACTTTACTCTGGGTTTTCG-3′AR5′-GAATTCTCAATGGTGATGGTGATGATGTTCACCTGTGTAGCAGATGGCGTCG-3′LPF5′-GGATCCAAGATGAAGAGTCTACTTCTTCTGGTG-3′LPR5′-GAATTCTCAATGGTGATGGTGATGATGGTTGTATGCTCTGAAGCAGTAGAC-3′LPFa5′-GGATCCTGATCATCTTTCAGACAACTATAC-3′HMSPF5′-GAATTCCTATAAATATGAAATTCTTAG-3′ Open table in a new tab Expression and Purification of Recombinant Proteins—Recombinant bacmids and baculoviruses were prepared, as described previously (8Shi S. Ciurli C. Cartman A. Pidoux I. Poole A.R. Zhang Y. Arthritis Rheum. 2003; 48: 2903-2915Crossref PubMed Scopus (45) Google Scholar), using the Bac-to-Bac Baculovirus Expression System, and the recombinant baculoviruses were used to infect High Five insect cells (Invitrogen) at a multiplicity of infection of 10. Recombinant proteins were purified from the culture supernatant by Sephadex G-25 and nickel-nitrilotriacetic acid-agarose (Qiagen, Mississauga, ON) column chromatography (8Shi S. Ciurli C. Cartman A. Pidoux I. Poole A.R. Zhang Y. Arthritis Rheum. 2003; 48: 2903-2915Crossref PubMed Scopus (45) Google Scholar). The concentrations of the recombinant proteins were determined by the method of Bradford (12Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214409) Google Scholar). Characterization of Purified Recombinant Proteins—To assess purity, recombinant proteins were analyzed by 12% SDS-PAGE under reducing conditions and silver staining and by Western blotting using specific monoclonal antibodies. The anti-His tag antibody was purchased from Qiagen. Antibody 12C5, specific for versican, 1C6 for aggrecan, and 8A4 for LP were provided by the Developmental Studies Hybridoma Bank (Department of Biological Sciences, the University of Iowa, Iowa City, IA). Antibody 12C5 recognizes an epitope located on the G1 domain of versican, and 1C6 recognizes a conserved sequence present once on each of the aggrecan G1 and G2 domains (13Fosang A.J. Hardingham T.E. Biochem. J. 1991; 273: 369-373Crossref PubMed Scopus (24) Google Scholar). To confirm further the authenticity of the recombinant proteins they were analyzed by 12% SDS-PAGE under reducing conditions and transferred onto a polyvinylidene difluoride membrane for N-terminal amino acid sequencing. Biotinylation of HA and Recombinant Proteins—Biotinylated HA was prepared by a modification of the method described by Melrose et al. (14Melrose J. Little C.B. Ghosh P. Anal. Biochem. 1998; 256: 149-157Crossref PubMed Scopus (17) Google Scholar), in which a reduced amount of biotinylation reagent was employed. In brief, 100 mg of HA (Sigma) was dissolved overnight at 4 °C in 20 ml of water. 0.8 g of adipic acid dihydrazide (Sigma) was added and the pH adjusted to 4.75 with 0.1 n HCl. 0.1 g of EDAC HCl (Sigma) was then added and the pH maintained at 4.75 by the addition of small aliquots of 0.1 n HCl. After incubation for 2 h at room temperature, the solution was adjusted to pH 7.0 with 0.1 n NaOH, dialyzed against water, and lyophilized to obtain hydrazido-HA. 100 mg of hydrazido-HA was redissolved overnight in 20 ml of 0.2 m NaHCO3 at 4 °C. 10 mg of biotin-3-sulfo-N-hydroxysuccinimide (Sigma) was added to the solution and incubation continued for 18 h at room temperature. Finally, 0.25 volume of 1 m Tris-HCl, pH 7.6, was added, and the product was dialyzed against PBS. In the original method of Melrose et al. (14Melrose J. Little C.B. Ghosh P. Anal. Biochem. 1998; 256: 149-157Crossref PubMed Scopus (17) Google Scholar), an approximately equimolar amount of biotinylation reagent, to HA in of HA In the present the biotinylation reagent was reduced to of that described by Melrose et al. that the acid be reduced to but not to Recombinant proteins were using according to the manufacturer's instructions. of VG1, LP, or HA in pH was added to and overnight at 4 °C. The were three with 20 and with and for 1 h at room temperature. After of protein at concentrations from to 4 in the was added to each well and for 2 h at room temperature. After the with in the was added and for 1 To the was added, and the was at in a of recombinant proteins or in and or were at °C for then onto membrane using a was used as a The membrane was for 1 h at room in 20 Tris-HCl, pH 7.6, 20 and and three with the membrane was with 1 LP in and for 2 h at room temperature. After was and LP was using an were at °C using a BIAcore system with a pH m and at a of 100 However, was used for and and were from Recombinant LP was on a using the of m and 0.2 m EDAC HCl was a for recombinant LP in 10 pH was onto a the amount of protein was Finally, of 0.1 m pH was the to the were generated using the by the protein with of HA to an 1 HA in was into a of an at a of the amount of the HA was The surface was also generated using the by the HA with concentrations of recombinant in the BIAcore were protein or surface for for by a for of a surface were for The surface was for by two of 100 of 10 HCl. The surface was for by two of 100 of HCl. Studies on the of VG1 and as LP with solution of VG1 or was with a solution of of the solution were and each solution was then HA on a or a The of VG1 or with LP to their on the HA surface was at 1 VG1, AG1, and LP at different concentrations and were a of cells for BIAcore were prepared using the generated using a surface were from the and the resulting were to were reduced to each of to of concentrations of protein the was by the to the binding and were determined from the The was as the The of the protein binding were prepared using the The of the by was by the of the at each by the as a function of is to The G1 domains of aggrecan (AG1) and versican and LP were expressed as recombinant proteins with their signal The recombinant proteins were 4 mg of recombinant VG1 and mg of were obtained of the the of LP was that it was to obtain for further To improve expression and the LP signal sequence was with the honeybee melittin signal This to of of The of the recombinant proteins was by SDS-PAGE and silver The recombinant proteins were also by Western blotting using a of N-terminal showed that recombinant VG1 has a N-terminal sequence to that for the mature core that the recombinant preparation two N-terminal and the mature core protein and two residues the two LP the component an N-terminal sequence which to the protein plus two residues These two residues were by the of the LP signal peptide with the honeybee melittin signal The component an N-terminal sequence with of the mature The of the three recombinant proteins on SDS-PAGE are than for the proteins and are with which is known to in insect To whether VG1 binds to LP and HA in a similar to AG1, an was LP or HA was and the to bind VG1 or was LP and HA VG1 and in a In binding of VG1 or to was The was VG1 or or HA was and the binding of LP was VG1 and both LP in a The of LP to bind VG1 to be than for for VG1 for and the binding affinity of VG1 for HA was than that of for VG1 for The that the incubation of VG1 and with the binding to LP A reverse that the incubation of LP with also its binding to VG1 or not These results demonstrate that disulfide in VG1 and and LP are for further the binding of VG1 and to LP, of and VG1 with LP were analyzed by In a BIAcore one of the binding is in the of a and the other is that the is a of the in BIAcore as it binds to the an in the of the at the and this is in The signal is to the of the This to the of the The solution is then by and of the surface is These a and from a of at different concentrations it is to In the recombinant VG1 or were recombinant LP were at a of 100 to of VG1 or a surface on which protein was were as a 4 of of the binding of LP to VG1 and to of of different concentrations of VG1 or the of cells LP or The (Table that the of VG1 with LP is than with LP, and the are similar for both VG1 and AG1. This in the for VG1 than for and and for the of VG1 and with link protein and VG1, AG1, and link protein with protein Link 0.2 Open table in a new tab HA HA was onto an A surface was generated by the but HA with of of the binding of VG1 to HA to HA and LP to HA The (Table that the of VG1 with HA was than and than LP, and of LP from HA was than VG1 and than AG1. This in the for LP the and for the The results obtained for the interaction of VG1 binding to HA were also analyzed by not The the to be which is in with the determined by using a (Table the of LP to the interaction of versican and aggrecan with HA, VG1 or that been with LP was were compared with from the of VG1, AG1, and LP In A and 1 the of VG1 or 2 the of the of VG1 with LP or with LP 4 the of 1 plus The between and 4 the of between VG1 and LP on the binding of both to VG1, the between and 4 that LP stabilizes the interaction of VG1 and AG1, the between and 4 was both the and that LP both the and the of is that under the conditions used in this study, LP not the of VG1 or with a is The baculovirus expression system used in this of VG1 and in the of LP was in the their signal A of LP in this expression system has been previously J. P.J. Biochem. J. PubMed Scopus Google Scholar). of the LP was obtained an insect signal peptide was used to the The of the insect signal peptide has been previously for other proteins (11Tessier D.C. Thomas D.Y. Khouri H.E. Laliberte F. Vernet T. Gene (Amst.). 1991; 98: 177-183Crossref PubMed Scopus (248) Google Scholar). The recombinant VG1 and were as proteins by N-terminal sequence and on the LP showed of at its The is the as that generated by P.J. Biochem. J. 1989; PubMed Scopus Google Scholar), which have not been to the G1 domain of aggrecan or is not in the insect cells which is responsible for this However, is that N-terminal of LP its to with G1 domains or was also that the insect cells generated G1 and LP disulfide that are for their as in the proteins J. Biol. Chem. Full Text PDF PubMed Google Scholar), and that they are recombinant proteins used for were analyzed at concentrations of less than to the of with the This is of in the of LP in which results in has been S. T. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). In the recombinant proteins were all at the concentrations BIAcore was used to assess the interaction of LP with LP on a was for interaction not has been previously that the aggrecan G1 domain and LP can with each other and with HA J. PubMed Scopus Google B. J. Biochem. Res. PubMed Scopus Google Scholar). it was that the of results in the of a stable ternary complex J. Biol. Chem. Full Text PDF PubMed Google M. M. Hardingham T.E. J. Biochem. J. PubMed Scopus Google Scholar). The present that the interaction of VG1 with LP or HA is higher affinity than that of the AG1. In both this to be by a higher of was also that of all three proteins LP has the affinity binding for HA, which is the of its of BIAcore also that the for LP binding to HA was than that for binding to HA Kimata K. Yamada Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These results are in with the of the interaction to pH from to in to the binding for interaction the pH S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In using and chromatography determined for and to be in the M. Biochem. J. PubMed Scopus Google Hardingham T.E. Biochem. J. PubMed Scopus Google Scholar), which well with the present a has also compared the interaction of recombinant VG1 and with LP and HA K. M. M. K. T. Kimata K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Their for the for the interaction of VG1, AG1, and LP with HA are all in the and as in the of the interaction with HA in the LP VG1 AG1. However, their results for the interaction of VG1 and with LP are different from in the present Although both determined a similar for the interaction with LP, in we that the affinity of VG1 for LP was higher than that of AG1, et al. K. M. M. K. T. Kimata K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) that the affinity of VG1 for LP is less than that for AG1. are for this including the of different for BIAcore and expression of recombinant proteins from different The used to LP binding in the present most of the binding with the of the and was to be a system. could also from in recombinant protein resulting from the of expression In this one that the most the in the binding is to that the for the interaction obtained with the insect system in the present was that that obtained with the human system. is to whether system the as this may with is from the present that the LP stabilization of VG1 or interaction with HA are not under the conditions of BIAcore analysis. This is not for interaction under the conditions of present the of BIAcore all degree of The of in is with that in the by et al. K. M. M. K. T. Kimata K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). first this to of proteoglycan stabilization by LP HA (5Hardingham T.E. Biochem. J. 1979; 177: 237-247Crossref PubMed Scopus (232) Google J. Biol. Chem. Full Text PDF PubMed Google A. S. Biochem. J. PubMed Scopus Google Scholar). This could be by the high of BIAcore for of The in the of LP to with the versican and aggrecan G1 domains and their the of whether LP was to function with both This is of at the present of the human sequence has the of of the LP with each LP gene to one of the of the aggrecan family of proteoglycans versican, and A. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). the gene the cartilage LP used in this to the versican gene than that of aggrecan is by the most abundant aggregating proteoglycan in is that the cartilage LP is in to with versican this proteoglycan and LP are present at in most tissues. This may the higher affinity of LP for this present is for the of the LP that the aggrecan gene in cartilage, and is on its to with the different the LP gene that the brevican gene on 1 is expressed in S. T. T. J. Y. Biochem. Res. 2000; PubMed Scopus Google Scholar), brevican is located K. M. Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The present that are in the of the cartilage LP to with the versican and aggrecan G1 domains and the this in to be of it is that the G1 domains in a similar to their and that other of the not In tissue concentrations of the proteoglycans can be higher than used in this in in the of aggrecan which is present in the in human articular it is not whether the between G1 at the higher Although further be to the of it is that the versican and aggrecan G1 domains were not to be under all We and for in preparation of the recombinant proteins and and for the