Monoclonal antibodies recognizing protease-generated neoepitopes from cartilage proteoglycan degradation. Application to studies of human link protein cleavage by stromelysin.
Monoclonal antibodies were raised that specifically recognize the NH2-terminal neoepitope sequence present in link protein cleavage products derived from stromelysin-degraded proteoglycan aggregate. Competitive enzyme-linked immunosorbent assay, using synthetic peptides as inhibitors, showed that one of these antibodies (CH-3) required, for antibody recognition, the free NH2-terminal amino acid isoleucine (residue 17 of the intact protein) in the sequence NH2-IQAENG at the stromelysin cleavage site of link protein 3. Human proteoglycan aggregate was digested with recombinant human stromelysin, bovine chymotrypsin, bovine trypsin, and porcine elastase, and their respective link protein degradation products were tested for immunoreactivity with antibody CH-3. Only stromelysin- and chymotrypsin-generated link protein 3 were recognized by antibody CH-3. Both of these enzymes generate link protein NH2 termini with the sequence 17IQAENG. . .; hence these studies indicated that monoclonal antibody CH-3 recognized this neoepitope sequence in only specific proteolytically modified link protein molecules. Since the occurrence of link protein 3 increases with aging, the incidence of CH-3 epitope in proteoglycans isolated from human knee articular cartilage of individuals of different ages was investigated. The prevalence of CH-3 epitope was found to be highest in newborn and adolescent articular cartilage samples. However, little CH-3 epitope was detected in older adult cartilage, although considerably more link protein 3 was present in these samples. These results suggest that additional proteolytic agents are responsible for the increased occurrence of link protein degradation products with aging.
Hughes
C E
CE
Division of Orthopedic Surgery, University of North Carolina, Chapel Hill 27599.
Caterson
B
B
White
R J
RJ
Roughley
P J
PJ
Mort
J S
JS
D000328:Adult; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002358:Cartilage, Articular / Q000737:chemistry*; D002648:Child; D002675:Child, Preschool; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D016326:Extracellular Matrix Proteins; D006801:Humans; D007223:Infant; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000378:metabolism*; D008875:Middle Aged; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology; D011506:Proteins / Q000276:immunology / Q000378:metabolism*; D011509:Proteoglycans / Q000378:metabolism*; D000099249:Proteoglycan Link Protein
D000911:Antibodies, Monoclonal; D000939:Epitopes; D016326:Extracellular Matrix Proteins; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases; D009363:Neoplasm Proteins; D009842:Oligopeptides; D011506:Proteins; D011509:Proteoglycans; D000099249:Proteoglycan Link Protein