Mammalian expression of full-length bovine aggrecan and link protein: formation of recombinant proteoglycan aggregates and analysis of proteolytic cleavage by ADAMTS-4 and MMP-13.
Aggrecan, a large chondroitin sulfate (CS) and keratan sulfate (KS) proteoglycan, has not previously been expressed as a full-length recombinant molecule. To facilitate structure/function analysis, we have characterized recombinant bovine aggrecan (rbAgg) and link protein expressed in COS-7 cells. We demonstrate that C-terminally truncated rbAgg was not secreted. Gel filtration chromatography of rbAgg and isolated glycosaminoglycan (GAG) chains, and their susceptibility to chondroitinase ABC digestion indicate that the GAG chains are predominantly CS, which likely occupy fewer serine residues than native aggrecan. To confirm functionality, we determined that rbAgg bound hyaluronan and recombinant link protein to form proteoglycan aggregates. In addition, cleavage of rbAgg by ADAMTS-4 revealed that the p68 form of ADAMTS-4 preferentially cleaves within the CS-2 domain, whereas the p40 form only effectively cleaves within the interglobular domain (IGD). MMP-13 cleaved rbAgg within the IGD, but cleaved more rapidly at a site within the CS domains, suggesting a role in C-terminal processing of aggrecan. Our results demonstrate that recombinant aggrecan can be used for in vitro analyses of matrix protease-dependent degradation of aggrecan in the IGD and CS domains, and both recombinant aggrecan and link protein can be used to study the assembly of proteoglycan aggregates with hyaluronan.
Miwa
Hazuki E
HE
Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106, USA.
Gerken
Thomas A
TA
Huynh
Tru D
TD
Flory
David M
DM
Hering
Thomas M
TM
D051722:ADAM Proteins / Q000378:metabolism*; D000071121:ADAMTS4 Protein; D053674:Aggrecans; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002417:Cattle; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis*; D002850:Chromatography, Gel; D017364:Collagenases / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis*; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D037181:Lectins, C-Type / Q000096:biosynthesis*; D053509:Matrix Metalloproteinase 13; D011348:Procollagen N-Endopeptidase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis
D053674:Aggrecans; D011508:Chondroitin Sulfate Proteoglycans; D016326:Extracellular Matrix Proteins; D006025:Glycosaminoglycans; D037181:Lectins, C-Type; D011994:Recombinant Proteins; D051722:ADAM Proteins; D017364:Collagenases; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human