Antibody mapping and tissue localization of globular and cysteine-rich regions of perlecan domain III.
Perlecan is the best-characterized basement membrane heparan sulfate proteoglycan. It has a large (approximately 400 KD) core protein consisting of five distinct domains. Domain III, a centrally located domain, contains three globular domains separated by cysteine-rich epidermal growth factor (EGF)-like repeats. Domain III has overall homology with the N-terminus of the laminin alpha 1-chain. The aim of this study was to map a library of nine rat monoclonal antibodies (MAbs) against murine perlecan core protein, using recombinant whole Domain III and defined subdomains of Domain III. ELISA and Western blotting showed that six of the nine MAbs recognized Domain III of perlecan, three of them mapping to globular Subdomain IIIc, and the other three recognized epitopes within the cysteine-rich regions. All six MAbs stained every basement membrane of several mouse organs as well as some connective tissues, including cartilage. Therefore, several distinct epitopes of perlecan Domain III are present in most if not all basement membranes and are not obscured by intermolecular interactions. These precisely mapped antibodies may therefore be useful in understanding the function of perlecan and its core protein.
Couchman
J R
JR
Department of Cell Biology, University of Alabama at Birmingham 35294-0019, USA.
Ljubimov
A V
AV
Sthanam
M
M
Horchar
T
T
Hassell
J R
JR
D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D003062:Codon; D003315:Cornea / Q000166:cytology; D003545:Cysteine; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000737:chemistry; D000939:Epitopes / Q000032:analysis*; D005455:Fluorescent Antibody Technique; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000032:analysis* / Q000737:chemistry* / Q000276:immunology; D007668:Kidney / Q000166:cytology; D008099:Liver / Q000166:cytology; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000166:cytology; D016133:Polymerase Chain Reaction; D011509:Proteoglycans / Q000032:analysis* / Q000737:chemistry* / Q000276:immunology; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D015183:Restriction Mapping; D012867:Skin / Q000166:cytology; D013154:Spleen / Q000166:cytology
D000911:Antibodies, Monoclonal; D003062:Codon; D003545:Cysteine; D017931:DNA Primers; D004815:Epidermal Growth Factor; D000939:Epitopes; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D011509:Proteoglycans; D011994:Recombinant Proteins; C071927:perlecan