Widespread expression of perlecan proteoglycan in basement membranes and extracellular matrices of human tissues as detected by a novel monoclonal antibody against domain III and by in situ hybridization.
Perlecan, a multidomain heparan sulfate proteoglycan (PG), is an intrinsic component of basement membranes and extracellular matrices. We used a prokaryotic expression vector to generate fusion proteins encoding various domains of human perlecan protein core and these recombinant proteins were used as immunogens to produce mouse anti-human monoclonal antibodies (MAb). One MAb, designated 7B5, was characterized by Western blotting and ELISA and was shown to react specifically with the laminin-like region of perlecan (Domain III) but not with two other fusion proteins encoding Domain II or V. This perlecan epitope was detected by immunoenzymatic staining in the basement membranes of human tissues including pituitary gland, skin, breast, thymus, prostate, colon, liver, pancreas, spleen, heart, and lung. All vascular basement membranes tested contained this gene product. In addition, sinusoidal vessels of liver, spleen, lymph nodes, and pituitary gland expressed high levels of perlecan in the subendothelial region. In situ hybridization, using as probe the same human cDNA-encoding Domain III, localized perlecan mRNA to specific cell types within the tissues and demonstrated that in skin, perlecan appears to be synthesized exclusively by connective tissue cells in the dermal layer. The availability of MAb against precise regions of human perlecan will allow the investigation of this gene product in normal and diseased states.
Murdoch
A D
AD
Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia 19107.
Liu
B
B
Schwarting
R
R
Tuan
R S
RS
Iozzo
R V
RV
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001485:Basement Membrane / Q000378:metabolism*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D005109:Extracellular Matrix / Q000378:metabolism*; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007124:Immunoenzyme Techniques; D017403:In Situ Hybridization; D051379:Mice; D010957:Plasmids; D011509:Proteoglycans / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins
D000911:Antibodies, Monoclonal; D000939:Epitopes; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D011509:Proteoglycans; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C071927:perlecan