Proteins of the kidney microvillar membrane. Effects of monensin, vinblastine, swainsonine and glucosamine on the processing and assembly of endopeptidase-24.11 and dipeptidyl peptidase IV in pig kidney slices

Abstract

The effects of various inhibitors were studied on the biogenesis of endopeptidase-24.11 (EC 3.4.24.11) and dipeptidyl peptidase IV (EC 3.4.14.5) in slices of renal cortex, from piglets of the Yucatan strain, maintained in organ culture. These microvillar peptidases were synthesized within membrane compartments and underwent glycosylation to yield high-mannose and complex forms [the preceding paper, Stewart & Kenny (1984) Biochem. J. 224, 549-558]. Monensin caused very gross ultrastructural changes in the proximal-tubular cells, resulting from distension of the Golgi sacs. It blocked the processing of the high-mannose to the complex glycosylated forms of the peptidases and prevented their assembly in the microvillar membrane. Swainsonine, an inhibitor of alpha-mannosidase II, generated new 'hybrid' forms of the proteins, intermediate in Mr between the high-mannose and the complex forms, but did not prevent assembly of the hybrid forms in microvilli. Vinblastine, an agent that affects microtubules, delayed, but did not abolish, either the processing or the transport to microvilli. Glucosamine interfered with the initial glycosylation reactions and generated heterogeneous sets of partially glycosylated polypeptides of lower Mr than the high-mannose forms. These results are discussed in relation to the site and mechanism of glycosylation and the involvement of the Golgi complex and microtubules in the biogenesis of these membrane peptidases.

Inhibition of the post‐translational processing of microvillar hydrolases is associated with a specific decreased expression of sucrase‐isomaltase and an increased turnover of glucose in Caco‐2 cells treated with monensin

“…The present results confirm the relevance of this model for such studies as far as treatment of the cells with monensin results in the same inhibition of the conversion of the enzymes from their high-mannose to their complex form as demonstrated for sucrase-isomaltase and dipeptidylpeptidase IV in small intestinal cells [ 161 or for dipeptidylpeptidase IV in kidney cells [17].…”

“…Monensin, an ionophore which binds monovalent cations, has been shown in a variety of systems to block the processing of newly synthesized glycoproteins at the site of the Golgi complex [ 1 l-l 51. In normal small intestinal [ 161 and kidney cells [17] it inhibits the conversion of the highmannose to the complex form of microvillar hydrolases. We show here that treatment of Caco-2 cells with monensin results not only in modifications of the post-translational processing of the enzymes, as would be expected, but also, unexpectedly, in a specific decrease in expression of sucrase-isomaltase and in marked modifications of the turnover of glucose.…”

Rapid sequestration of DPP IV/CD26 and other cell surface proteins in an autophagic-like compartment in Caco-2 cells treated with forskolin

“…A powerful approach to a better understanding of this complex phenomenon is the use of drugs that may act on traf ficking and/or sorting events. This includes: (i) drugs known to inhibit glycosylation processing enzymes (Elbein, 1987;Ogier-Denis et al, 1990;Trugnan et al, 1991); (ii) drugs modifying structures and/or functions of subcellulav compart ments, as for example monensin (Akin et al, 1987;Stewart and Kenny, 1984;Tartakoff, 1983) or brefeldin A (Fujiwara et al, 1988;Lippincott-Schwartz et al, 1989;Low et al, 1992;Misumi et al, 1986;Pelham, 1991); and (iii) drugs acting on the cytoskeleton, especially on micro tubules (Achler et al, 1989;Eilers et al, 1989;Gilbert et al, 1991;Hunziker et al, 1991;Matter, 1990b).…”