Structural properties of recombinant domain III-3 of perlecan containing a globular domain inserted into an epidermal-growth-factor-like motif.
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 for 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.
Schulze
B
B
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Mann
K
K
Battistutta
R
R
Wiedemann
H
H
Timpl
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D004220:Disulfides / Q000737:chemistry; D010450:Endopeptidases / Q000737:chemistry; D004815:Epidermal Growth Factor / Q000737:chemistry*; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D006801:Humans; D007106:Immune Sera; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011487:Protein Conformation; D011509:Proteoglycans / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004220:Disulfides; D010450:Endopeptidases; D004815:Epidermal Growth Factor; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D007106:Immune Sera; D010446:Peptide Fragments; D011509:Proteoglycans; D011994:Recombinant Proteins; C071927:perlecan