A fragment comprising approximately domain III-3 of the basement membrane heparan sulfate proteoglycan perlecan was prepared in recombinant form from kidney cell clones. This fragment was predicted to contain a cysteine-free globular domain inserted within an epidermal-growth-factor(EGF)-like motif (L4 module) and three additional EGF-like motifs (LE module) without large inserts. This prediction was confirmed by electron microscopy, which demonstrated a globule joined to a very short rod-like segment. The globule was selectively destroyed by pepsin, which also demonstrated that its insertion into an EGF-like motif did not prevent the typical disulfide connections known far such motifs. Yet the globule was more stable against neutral proteinases. The fragment showed a distinct content (55-60%) of alpha helical and beta structure and a partially reversible melting of the conformation in 6 M guanidine. Antibodies raised against recombinant domain III-3 demonstrated a complete cross-reaction with tissue-derived perlecan but not with laminin and a distinct basement membrane staining of tissue sections. Most of the epitopes were lost after reduction and alkylation. Together the data demonstrated a proper folding of recombinant domain III-3 similar to its structure in the native protein and provided the first structural evidence for a novel globular protein motif L4 based on an EGF-like scaffold.
Structural-properties of Recombinant Domain Iii-3 of Perlecan Containing A Globular Domain Inserted Into An Epidermal-growth-factor-like Motif Rid C-4254-2008
BATTISTUTTA, ROBERTO;
1995
Abstract
A fragment comprising approximately domain III-3 of the basement membrane heparan sulfate proteoglycan perlecan was prepared in recombinant form from kidney cell clones. This fragment was predicted to contain a cysteine-free globular domain inserted within an epidermal-growth-factor(EGF)-like motif (L4 module) and three additional EGF-like motifs (LE module) without large inserts. This prediction was confirmed by electron microscopy, which demonstrated a globule joined to a very short rod-like segment. The globule was selectively destroyed by pepsin, which also demonstrated that its insertion into an EGF-like motif did not prevent the typical disulfide connections known far such motifs. Yet the globule was more stable against neutral proteinases. The fragment showed a distinct content (55-60%) of alpha helical and beta structure and a partially reversible melting of the conformation in 6 M guanidine. Antibodies raised against recombinant domain III-3 demonstrated a complete cross-reaction with tissue-derived perlecan but not with laminin and a distinct basement membrane staining of tissue sections. Most of the epitopes were lost after reduction and alkylation. Together the data demonstrated a proper folding of recombinant domain III-3 similar to its structure in the native protein and provided the first structural evidence for a novel globular protein motif L4 based on an EGF-like scaffold.
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