A plasma membrane antigen of rat glomerular epithelial cells. Antigenic determinants involving N-linked sugar residues in a 140-kilodalton sialoglycoprotein of the podocytes.
The aim of the present study was to investigate the antigenic make up of the plasma membrane of rat glomerular visceral epithelial cells (GEP). A crude plasma membrane fraction (PM) was extracted by 1% sodium dioxycholate from isolated rat glomeruli. PM was digested with neuraminidase (NRD) and purified by the Helix pomatia agglutinin (HPA)-affinity column. When studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the HPA-affinity purified plasma membrane fraction (HPA-PM) formed two bands, a main band of 160 kilodaltons (kd) and a smaller band of 40 kd. By Western blot analysis, the antibodies raised in a rabbit against HPA-PM (RbAHPA-PM) reacted only with the 160-kd protein of HPA-PM, or with the relevant 140-kd protein of PM when PM was digested by NRD. The 140-kd protein of PM was reactive with wheat germ agglutinin and, when treated with NRD, was reactive with HPA and peanut lectin. The 160-kd protein of HPA-PM was degraded by endoglycosidase-F and lost its reactivity with RbAHPA-PM. These results suggest that RbAHPA-PM react with antigenic sites involving N-linked sugar residues of a 140-kd sialoglycoprotein, presumably podocalyxin. Immunohistochemical studies using normal rat kidney tissues treated with NRD as substratum showed that RbAHPA-PM bound to the free surface of GEP but not with soles of the foot processes or with other structures of the kidney. In rats intravenously injected with NRD and subsequently with RbAHPA-PM, antibodies were rapidly fixed (within 1 hour) to the free surface of GEP. Immunofluorescence study showed that RbAHPA-PM also reacted with human glomeruli after treatment with NRD. These results suggest that GEP express surface sugar residues that are potential targets for direct immunologic attack.
Third Department of Internal Medicine, Nagoya University School of Medicine, Japan.
I
I
Ozaki
Y
Y
Ito
A
A
Fukatsu
N
N
Suzuki
F
F
Yoshida
Y
Y
Watanabe
N
N
Sakamoto
S
S
Matsuo
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000941:Antigens / Q000276:immunology; D002462:Cell Membrane / Q000276:immunology* / Q000648:ultrastructure; D000939:Epitopes; D005260:Female; D006801:Humans; D007678:Kidney Glomerulus / Q000166:cytology / Q000276:immunology* / Q000648:ultrastructure; D037102:Lectins / Q000276:immunology*; D008970:Molecular Weight; D009439:Neuraminidase; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D012795:Sialoglycoproteins / Q000276:immunology*
D000906:Antibodies; D000941:Antigens; D000939:Epitopes; C038095:Helix lectin; D037102:Lectins; D012795:Sialoglycoproteins; D009439:Neuraminidase