Characterization of Recombinant Perlecan Domain I and Its Substitution by Glycosaminoglycans and Oligosaccharides


R. Timpl, Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany


Recombinant mouse perlecan domain I (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 1 A) 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 8–10kDa 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 a helix (20%) and β structure (60%) in fragment IA, consistent with predictions of a novel SEA protein module located in the C-terminal part of domain I.


cyanogen bromide

SEA module

a protein module first identified in sperm protein, enterokinase and agrin


Chondroitinase ABC (EC


endoproteinase Asp-N (EC


endoproteinase Glu-C (EC


endoproteinase Lys-C (EC


heparitinase I (EC