N- and O-linked keratan sulfate on the hyaluronan binding region of aggrecan from mature and immature bovine cartilage.
In the hyaluronan binding region (HABR) peptide of aggrecan, there is a marked increase in the level of keratan sulfate (KS) during aging. To determine the sites of KS attachment, KS-containing peptides were prepared from HABRs from immature and mature bovine articular cartilage by digestion with trypsin or papain followed by carbohydrate analysis and peptide sequencing. KS is attached to Thr42 within loop A in mature, but not in immature, HABR. Within loop B KS is N-linked to Asn220 in both HABRs, but in the immature HABR the chains are shorter. Asn314 in loop B' of mature HABR is substituted either with a KS chain or with an oligosaccharide of the complex type. In immature HABR this site does not carry KS. In the interglobular domain, 2 threonine residues within the sequence TIQTVT are substituted in both calf and steer, and in steer further substitution occurs within the sequence NITEGEA, which contains a major catabolic cleavage site (Sandy, J., Neame, P.J., Boynton, R., and Flannery, C.R. (1991) J. Biol. Chem. 266, 8683-8685). The extreme polydispersity of mature HABR was investigated by preparing four subfractions of increasing molecular size which had essentially the same protein core, i.e. Val1-Arg367 or Val1-Arg375. The smaller species lacked the KS chains attached to loop A. These results show that KS substitution occurs within each of the disulfide-bonded loops of the HABR, that the KS may be either N- or O-linked, and that variations in the addition of KS are responsible for the polydispersity of mature HABR.
Barry
F P
FP
Shriners Hospital for Crippled Children, University of South Florida College of Medicine, Tampa 33612, USA.
Rosenberg
L C
LC
Gaw
J U
JU
Gaw
J U
JU
Koob
T J
TJ
Neame
P J
PJ
D053674:Aggrecans; D000375:Aging / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002241:Carbohydrates / Q000032:analysis; D002358:Cartilage, Articular / Q000254:growth & development / Q000378:metabolism*; D002417:Cattle; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism; D002846:Chromatography, Affinity; D016326:Extracellular Matrix Proteins; D006820:Hyaluronic Acid / Q000302:isolation & purification / Q000378:metabolism*; D007632:Keratan Sulfate / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D037181:Lectins, C-Type; D008297:Male; D008969:Molecular Sequence Data; D009919:Orchiectomy; D010206:Papain; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D011509:Proteoglycans / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014357:Trypsin
D053674:Aggrecans; D002241:Carbohydrates; D011508:Chondroitin Sulfate Proteoglycans; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D010446:Peptide Fragments; D011509:Proteoglycans; D006820:Hyaluronic Acid; D007632:Keratan Sulfate; D014357:Trypsin; D010206:Papain