Development of monoclonal antibodies against different protein and carbohydrate epitopes of dipeptidyl peptidase IV from rat liver plasma membranes

Abstract

Dipeptidyl peptidase IV (DPP IV) is a serine exopeptidase expressed at high levels in rat kidney, liver and lung. We established eight monoclonal antibodies against partially purified DPP IV from rat liver plasma membranes. By means of a competitive dot blot assay with purified DPP IV, these antibodies were shown to recognize four different epitopes of the glycoprotein, designated A -D. The epitopes are located on the extracellular domain of DPP IV, as shown by papain digestion of liver plasma membranes. Treatment of DPP IV with neuraminidase and glycopeptide N-glycosidase F, as well as incubation of heptocytes with the a-mannosidase I inhibitor deoxymannojirimycin, revealed that epitope A may be formed by a mannose-rich sugar chain and epitope D might represent a complex carbohydrate structure in the mature glycoprotein, while the epitopes B and C are formed by the protein moiety. Concanavalin A reduced the binding of monoclonal antibody to epitope A by 78%. Binding to epitope D was blocked by 73% with wheat germ lectin, and by more than 99% with sialic acid; epitopes B and C were unaffected by any of the lectins or sugars tested. The immunological cross-reactivity with DPP IV from Morris hepatoma 7777 was demonstrated with monoclonal antibodies against epitopes A -C. Epitope D was not recognized on hepatoma DPP IV. However, in addition to DPP IV, four hepatoma plasma membrane glycoproteins were precipitated by the monoclonal antibody against the epitope D, indicating that this epitope is not uniquely restricted to DPP IV.

Functional Organization of the Golgi Apparatus in Glycosphingolipid Biosynthesis

“…t-LacCer was characterized and quantified as described (39). Immunological quantification of DPP IV as a marker for the apical domains of liver plasma membranes (B) (48,49), rab5 as a marker for early endosomes (C) (50,51), and TGN38 as a marker for TGN (D) (52). Relative immunoactivity in % in B-D equals percentage of pixels in each individual lane relative to the pixels summarized from all lanes.…”

“…Of each 1-ml fraction, the following aliquots were used: 10 l for esterase, 20 l for Gal-T, and 20 l for GlcNAc-P-T. In addition, various types of membranes were analyzed immunologically with antibodies directed against dipeptidylpeptidase IV (DPP IV) (rat liver plasma membranes (48,49)), rab5 (early endosomes (50, 51)), and TGN38 (trans Golgi network (52)). Several independent gradients were run with nearly the same results.…”

“…2A with a peak in fraction 18. The following marker proteins were quantitated immunologically to determine membrane contaminants within the fractions across the gradient: for liver plasmamembranes DPP IV (48,49) (Fig. 2B), for early endosomal membranes rab5 (50,51) (Fig.…”

Biosynthesis and metabolism of dipeptidylpeptidase IV in primary cultured rat hepatocytes and Morris hepatoma 7777 cells

“…During internalization and surface recycling, oligosaccharide chains of DPP IV have been shown to undergo continuous loss and re-transfer of outer L-fucose and sialic acid residues thought to represent trimming by cellular glycosidases and a possible repair mechanism [29,30,331. Furthermore, as evidenced by use of a monoclonal antibody against a carbohydrate epitope, glycosylation of DPP IV undergoes distinct alterations during transformation of hepatocytes that parallel changes in the expression in hepatoma cells and hepatoma tissues [34].…”

Chemical cross‐linking leads to two high molecular mass aggregates of rat α1β1 integrin differing in their conformation but not in their composition

“…The antiserum against rat a9 integrin subunit was kindly provided by Dr. Staffan Johansson [12]. mAb 13.4 is specific for rat dipeptidylpeptidase IV [13].…”