Two-dimensional peptide mapping of cross-linked type IX collagen in human cartilage.
Type IX collagen is a quantitatively minor component of hyaline cartilage that is essential for the normal structural integrity of the tissue. Purification and analysis are difficult because the mature protein is insoluble as a cross-linked integral component of the fibrillar matrix. In order to view a peptide map of the total pool of type IX collagen in a cartilage sample, a selective method based on Western blot analysis was developed for displaying collagen IX peptides in a cyanogen bromide digest of tissue. Digests were partially resolved by reverse-phase HPLC, individual fractions were run on SDS-PAGE and then transblotted to membrane, and the collagen IX fragments were revealed using an anti-collagen IX rabbit antiserum. All major CB-peptides from alpha1(IX), alpha2(IX), and alpha3(IX) chains in the resulting two-dimensional display were identified by amino-terminal sequence analysis. Cross-linked peptides originating from sites of covalent interaction between collagen types IX and II and between IX and IX were also defined. By comparison with an analysis of soluble type IX collagen from chondrocyte culture medium, the results showed that the pool of type IX collagen molecules in fetal and adult human cartilage is extensively cross-linked intermolecularly at sites previously revealed by other methods using purified protein. This sensitive, direct method has the potential to screen for abnormalities in the content and properties of type IX collagen in tissue samples, for example, in the study of heritable chondrodysplasia syndromes and the pathogenesis of cartilage destruction in osteoarthritis.
Ichimura
S
S
Orthopaedic Research Laboratories, University of Washington, Seattle 98195, USA.
Wu
J J
JJ
Eyre
D R
DR
D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002356:Cartilage / Q000737:chemistry*; D003094:Collagen / Q000737:chemistry* / Q000235:genetics; D003432:Cross-Linking Reagents; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010449:Peptide Mapping / Q000379:methods; D011817:Rabbits
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D003094:Collagen