Regular Article
Localization of Glycosaminoglycan Substitution Sites on Domain V of Mouse Perlecan

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Abstract

Perlecan, the predominant basement membrane proteoglycan, has previously been shown to contain glycosaminoglycans attached at serine residues, numbers 65, 71, and 76, in domain I. However, the C-terminal domains IV and V of this molecule may also be substituted with glycosaminoglycan chains, but the exact substitution sites were not identified. The amino acid sequence of mouse perlecan reveals many ser-gly sequences in these domains that are possible sites for glycosaminoglycan substitution. We expressed recombinant domain IV and/or V of mouse perlecan in COS-7 cells and analyzed glycosaminoglycan substitution. Both heparan sulfate and chondroitin sulfate chains could be detected on recombinant domain V. One site, ser-gly-glu (serine residue 3593), toward the C-terminal region of domain V is a substitution site for heparan sulfate. When this sequence was absent, chondroitin/dermatan sulfate substitution was deleted, and the likely site for this galactosaminoglycan substitution was ser-gly-ala-gly (serine residue 3250) on domain V.

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    Abbreviations used: GAG, glycosaminoglycan; HS, heparan sulfate; CS, chondroitin sulfate; DS, dermatan sulfate; LDL, low density lipoprotein; N-CAM, neural cell adhesion molecule; EGF, epidermal growth factor; ESH, Engelbreth–Holm–Swarm; DMEM, Dulbecco's modified Eagle's medium; DW, distilled water; PBS, Dulbecco's phosphate-buffered saline; TBS, Tris-buffered saline; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; FGF, fibroblast growth factor.

    1

    To whom correspondence and reprint requests should be addressed at Department of Cell Biology, University of Alabama at Birmingham, VH 201 C University Boulevard, Birmingham, AL 35294-0019. Fax: 205-975-9956. E-mail: [email protected].

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