Hyaluronan-binding region of aggrecan from pig laryngeal cartilage. Amino acid sequence, analysis of N-linked oligosaccharides and location of the keratan sulphate.
The hyaluronan-binding region (HABR) was prepared from pig laryngeal cartilage aggrecan and the amino acid sequence was determined. The HABR had two N-termini: one N-terminal sequence was Val-Glu-Val-Ser-Glu-Pro (367 amino acids in total), and a second N-terminal sequence (Ala-Ile-Ser-Val-Glu-Val; 370 amino acids in total) was found to arise due to alternate cleavage by the signal peptidase. The N-linked oligosaccharides were analysed by examining their reactivity with a series of lectins. It was found that the N-linked oligosaccharide on loop A was of the mannose type, while that on loop B was of the complex type. No reactivity was detected between the N-linked oligosaccharide on loop B' and any of the lectins. The location of keratan sulphate (KS) in the HABR was determined by Edman degradation of the immobilized KS-containing peptide. The released amino acid derivatives were collected and tested for the presence of epitope to antibody 5-D-4. On the basis of 5-D-4 reactivity and sequencing yields, the KS chains are attached to threonine residues 352 and 357. There is no KS at threonine-355. This site is not in fact in G1, but about 16 amino acid residues into the interglobular domain. Comparison of the structure of the KS chain from the HABR and from the KS domain of pig laryngeal cartilage aggrecan was made by separation on polyacrylamide gels of the oligosaccharides arising from digestion with keratanase. Comparison of the oligosaccharide maps suggests that the KS chains from both parts of the aggrecan molecule have the same structure.
Barry
F P
FP
Shriners' Hospital for Crippled Children, Tampa, FL 33612-9499.
Gaw
J U
JU
Young
C N
CN
Neame
P J
PJ
D053674:Aggrecans; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002356:Cartilage / Q000378:metabolism*; D011508:Chondroitin Sulfate Proteoglycans / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins; D006820:Hyaluronic Acid / Q000378:metabolism*; D007632:Keratan Sulfate / Q000032:analysis; D007830:Larynx / Q000378:metabolism*; D037181:Lectins, C-Type; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis; D010449:Peptide Mapping; D011509:Proteoglycans / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013552:Swine; D014357:Trypsin
D053674:Aggrecans; D011508:Chondroitin Sulfate Proteoglycans; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D009844:Oligosaccharides; D011509:Proteoglycans; D006820:Hyaluronic Acid; D007632:Keratan Sulfate; D014357:Trypsin