Monoclonal antibodies as probes for determining the microheterogeneity of the link proteins of cartilage proteoglycan.

B Caterson,J R Baker,J E Christner,Y Lee,M Lentz

doi:10.1016/s0021-9258(17)39186-x

Abstract

Monoclonal antibodies were raised against Swarm rat chondrosarcoma link protein 2. Two of the resultant hybridomas (9/30/6-A-1 and 9/30/8-A-4) were used in structural analyses of the link proteins. The 9/30/6-A-1 monoclonal antibody recognized an epitope which was only present on rat chondrosarcoma link protein 2. This epitope was absent in rat chondrosarcoma link protein 3 obtained after trypsin or clostripain treatment of rat chondrosarcoma proteoglycan aggregate, indicating that proteolytic digestion either removed or modified the epitope. Contrasting this, the 9/30/8-A-4 monoclonal antibody recognized an epitope present in link protein(s) 1, 2, or 3 isolated from cartilage of several animal species (rat, bovine, human, and chicken). Rat chondrosarcoma link protein 2 was digested with Staphylococcus aureus V8 protease, and the resulting peptides were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to immunolocation analyses. The 9/30/6-A-1 and 9/30/8-A-4 monoclonal antibodies recognized epitopes in two different halves of the link protein molecule. The 9/30/8-A-4 monoclonal antibody was used to identify proteolytic cleavage peptides common to the individual link proteins (1, 2, or 3) purified from cartilage proteoglycans of several animal species. Digestion of rat chondrosarcoma link protein 2 with endoglycosidase H or alpha-mannosidase increased its electrophoretic mobility to that of link protein 3 and removed or altered the determinant recognized by the 9/30/6-A-1 monoclonal antibody, indicating that a high-mannose oligosaccharide chain was part of the antigenic determinant. The 9/30/8-A-4 monoclonal recognition of epitope was unaffected by endo- or exoglycosidase treatment. Endo- and exoglycosidase treatment of bovine nasal cartilage link proteins also altered their electrophoretic mobility, indicating that high-mannose oligosaccharide structures on the various link proteins (1, 2, or 3) accounted for the microheterogeneity observed in sodium dodecyl sulfate-polyacrylamide gels.

Highlights

Monoclonal antibodies were raised against Swarm traction and isolation have been recently reviewed [5,6]
This epitope was absent in rat chondrosar- Within the proteoglycan aggregate structure, the link procoma link protein 3 obtained after trypsin or clostripain treatment of rat chondrosarcoma proteoglycan aggregate, indicating that proteolytic digestion either removed or modified the epitope
Digested withStaphylococcus aureus V8 protease, and Three link proteins havebeenidentifiedinbovine nasal the resulting peptides were fractionated by sodium cartilage proteoglycan aggregate: link protein 1, link protein dodecyl sulfate-polyacrylamide gel electrophoreasnisd 2, and a minorcomponent link protein 3, with molecular subjected to immunolocation analyses

Summary

Introduction

Monoclonal antibodies were raised against Swarm traction and isolation have been recently reviewed [5,6]. Isolated link proteins (1or 2 ) from rat chondrosarcoma, bovine nasal cartilage, and chick sternal cartilage proteoglycan were digested with 2.5 pg of S. aureus V8 protease, and the peptides were separated by electrophoresis, electrophoretically transferred to nitrocellulose, and identified by immunolocation using the '251-labeled913018-A4 monoclonal antibody.

Results

Conclusion