Novel Monoclonal Antibody mAbG3A5Recognizes 138-kDa Glyocoprotein Localized on the Golgi Membrane

Abstract

Partially purified Golgi membranes of HeLa cells were used as antigen to produce a novel monoclonal antibody (mAb G3A5). The mAb G3A5 specifically labeled Golgi apparatus of human and monkey cultured cells as ascertained by indirect immunofluorescence but did not stain those of bovine or mouse cells. Treatment with nocodazole and brefeldin A (BFA) induced fragmentation and redistribution of the staining. Western immunoblot analysis showed that mAb G3A5 was directed against a single polypeptide with an apparent molecular mass of 138-kDa (p138 antigen). The p138 antigen is an integral membrane protein of the Golgi apparatus, as assessed by several assays: protease protection, salt wash and flotation in sucrose density gradient centrifugation. The p138 antigen was purified using immunoaffinity chromatography. The apparent molecular mass of the p138 antigen decreased by 2 to 4 kDa after treatment with the peptide: N-glycosidase F, while digestion with ENDO F or Neuraminidase did not have this effect. Thus, p138 antigen is a glycoprotein containing asparagine linked carbohydrates.