Characterization of recombinant perlecan domain I and its substitution by glycosaminoglycans and oligosaccharides.
Recombinant mouse perlecan domain 1(173 residues) was produced in transfected embryonic kidney cells and purified from the culture medium on DEAE-cellulose. It was shown to be modified by glycosaminoglycans and could be partially separated into two protein pools which were either substituted with heparan sulfate (fragment IA) or, to a smaller extent (20%), with chondroitin/dermatan sulfate or a mixture of both glycosaminoglycans (fragment IB). The average molecular mass of the glycosaminoglycans was about 8-10 kDa and, thus, smaller than in tissue-derived perlecans. Sequence and carbohydrate analyses localized the heparan sulfate attachment site to three Ser residues within SGD consensus sequences. Furthermore, the N-terminal part of fragment IA contained six Thr/Ser residues substituted by branched galactosamine-containing oligosaccharides and an N-substituted Asn residue. Fragment I was also shown to contain unique immunological epitopes which are not dependent on glycosaminoglycans and are shared by tissue-derived perlecan. Circular dichroism demonstrated a distinct alpha helix (20%) and beta structure (60%) in fragment IA, consistent with predictions of a novel SEA protein module located in the C-terminal part of domain I.
Costell
M
M
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Mann
K
K
Yamada
Y
Y
Timpl
R
R
D000595:Amino Acid Sequence; D000818:Animals; D002809:Chondroitin Sulfates / Q000737:chemistry; D002942:Circular Dichroism; D018604:Epitope Mapping; D006025:Glycosaminoglycans / Q000737:chemistry*; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000737:chemistry*; D051379:Mice; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D011487:Protein Conformation; D011509:Proteoglycans / Q000737:chemistry*; D011994:Recombinant Proteins
D002809:Chondroitin Sulfates; D006025:Glycosaminoglycans; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D009844:Oligosaccharides; D011509:Proteoglycans; D011994:Recombinant Proteins; C071927:perlecan