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Biosynthesis of intestinal microvillar proteins. Processing of aminopeptidase N by microsomal membranes.

ABSTRACT: The biosynthesis of small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in a cell-free translation system derived from rabbit reticulocytes. When dog pancreatic microsomal fractions were present during translation, most of the aminopeptidase N synthesized was found in a membrane-bound rather than a soluble form, indicating that synthesis of the enzyme takes place on ribosomes attached to the rough endoplasmic reticulum. The microsomal fractions process the Mr-115 000 polypeptide, which is the primary translation product of aminopeptidase N, to a polypeptide of Mr 140 000. This was found to be sensitive to the action of endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96), showing that aminopeptidase N undergoes transmembrane glycosylation during synthesis. The position of the signal sequence in aminopeptidase N was determined by a synchronized translation experiment. It was found that microsomal fractions should be added before about 25% of the polypeptide was synthesized to ensure processing to the high-mannose glycosylated form. This suggests that the signal sequence is situated in the N-terminal part of the aminopeptidase N. The size of the cell-free translation product in the absence of microsomal fractions was found to be similar to that on one of the forms of the enzyme obtained from tunicamycin-treated organ-cultured intestinal explants.

SUBMITTER: Danielsen EM

PROVIDER: S-EPMC1152024 | biostudies-other | 1983 Apr

REPOSITORIES: biostudies-other

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Biosynthesis of intestinal microvillar proteins. The effect of swainsonine on post-translational processing of aminopeptidase N.

Project description:The post-translational processing of pig small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in organ-cultured mucosal explants. Exposure of the explants to swainsonine, an inhibitor of Golgi mannosidase II, resulted in the formation of a Mr-160000 polypeptide, still sensitive to endo-beta-N-acetylglucosaminidase H. Swainsonine caused only a moderate inhibition of transport of the enzyme through the Golgi complex and the subsequent expression in the microvillar membrane. This may imply that the trimming of the high-mannose core and complex glycosylation of N-linked oligosaccharides is not essential for the transport of aminopeptidase N to its final destination. A different type of processing was observed to take place in the presence of swainsonine, resulting in a considerable increase in apparent Mr (from 140000 to 160000). This processing could not be ascribed to N-linked glycosylation, since treatment of the Mr-160000 polypeptide with endo-beta-N-acetylglucosaminidase H only decreased its apparent Mr by 15000. The susceptibility of the mature Mr-166000 polypeptide, but not the Mr-140000 polypeptide, to mild alkaline hydrolysis suggests that aminopeptidase N becomes glycosylated with O-linked oligosaccharides during its passage through the Golgi complex. Aminopeptidase N was not labelled by [3H]palmitic acid, indicating that the processing of the enzyme does not include acylation.

| S-EPMC1152508 | biostudies-other

Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on aminopeptidase N and sucrase-isomaltase.

Project description:The biogenesis of two microvillar enzymes, aminopeptidase N (EC 3.4.11.2) and sucrase (EC 3.2.1.48)-isomaltase (EC 3.2.1.10), was studied by pulse-chase labelling of pig small-intestinal explants kept in organ culture. Both enzymes became inserted into the membrane during or immediately after polypeptide synthesis, indicating that translation takes place on ribosomes attached to the rough endoplasmic reticulum. The earliest detectable forms of aminopeptidase and sucrase-isomaltase were polypeptides of Mr 140 000 and 240 000 respectively. These polypeptides were susceptible to treatment with endo-beta-N-acetylglucosaminidiase H (EC 3.2.1.96), suggesting that the microvillar enzymes during or immediately after completion of protein synthesis become glycosylated with a 'high-mannose' oligosaccharide structure similarly to other plasma-membrane and secretory proteins. After 20--40 min or 60--90 min of chase, respectively, aminopeptidase N and sucrase-isomaltase were reglycosylated to give the polypeptides of Mr 166 000 (aminopeptidase N) and 265 000 (sucrase-isomaltase). These were expressed at the microvillar membrane after 60--90 min. During the entire process of synthesis and transport to the microvillar membrane the enzymes were bound to membranes, indicating that the biogenesis of aminopeptidase N and sucrase-isomaltase occurs in accordance with the membrane flow hypothesis.

| S-EPMC1158402 | biostudies-other

Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV.

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| S-EPMC1154236 | biostudies-other

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| S-EPMC1147486 | biostudies-other

Biosynthesis of intestinal microvillar proteins. Translational evidence in vitro that aminopeptidase N is synthesized as a Mr-115000 polypeptide.

Project description:A crude RNA fraction, prepared from pig small intestine, was found to be more efficient than a fraction enriched in polyadenylated RNA in directing translation of polypeptides with Mr greater than 100000 in a rabbit reticulocyte lysate system. Aminopeptidase N (EC 3.4.11.2) synthesized in vitro was immunopurified from the translation mixture and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It was found to have an apparent Mr of 115000 regardless of whether the translation was performed in the absence or presence of proteinase inhibitors. This result contradicts the possibility of aminopeptidase N being synthesized as a large single-chain precursor polypeptide.

| S-EPMC1158348 | biostudies-other

Biosynthesis of intestinal microvillar proteins. Role of the Golgi complex and microtubules.

Project description:The effect of monensin and colchicine on the biogenesis of aminopeptidase N (EC 3.4.11.2), aminopeptidase A (EC 3.4.11.7), dipeptidyl peptidase IV (EC 3.4.14.5), sucrase (EC 3.2.1.48)-isomaltase (EC 3.2.1.10) and maltase-glucoamylase (EC 3.2.1.20) was studied in organ-cultured pig small-intestinal explants. On the ultrastructural level, monensin (1 microM) caused an increasingly extensive dilation and vacuolization of the Golgi complex during 4h exposure of the explants. On the molecular level, the effect of monensin was twofold. (1) The processing from the initial high-mannose-glycosylated form to the mature complex-glycosylated form was arrested. For some of the enzymes studied, intermediate stages between the high-mannose and complex forms could be seen, probably corresponding to 'trimmed' or partially complex-glycosylated polypeptides. (2) Labelled microvillar enzymes failed to reach their final destination. These findings suggest the involvement of the Golgi complex in the post-translational processing and transport of microvillar enzymes. The presence in the growth medium of colchicine (50 micrograms/ml) caused a significant inhibition of the appearance of newly synthesized enzymes in the microvillar membrane during a 3 h labelling period. Since synthesis and post-translational modification of the microvillar enzymes were largely unaffected by colchicine, the results obtained suggest that microtubules play a role in the final transport of the enzymes from the Golgi complex to the microvillar membrane.

| S-EPMC1152467 | biostudies-other

Biosynthesis of intestinal microvillar proteins. Characterization of intestinal explants in organ culture and evidence for the existence of pro-forms of the microvillar enzymes.

Project description:Explants of pig small intestine were maintained at 37 degrees C in organ culture for periods up to 24 h in a system using Trowell T-8 medium supplemented with 10% foetal-calf serum. The mucosal morphology was well preserved during culture, as judged by light and electron microscopy. The explant contents of protein and two brush-border enzymes, microvillus aminopeptidase (EC 3.4.11.2) and dipeptidyl peptidase IV (EC 3.4.14.5), were not significantly modified during culture compared with controls, but a moderate, continuous release of both protein and enzyme activities into the medium was observed. Continuous labelling with [35S]methionine resulted in an even incorporation of radioactivity in the protein components, and the rate of labelling only moderately decreased over the 24 h period. The polypeptide compositions of sucrase (EC 3.2.1.48)--isomaltase (EC 3.2.1.10), maltase--glucoamylase (EC 3.2.1.20) lactase (EC 3.2.1.23)--phlorizin hydrolase (EC 3.2.1.62), microvillus aminopeptidase and aspartate aminopeptidase (EC 3.4.11.7) synthesized during culture were studied, and some were found to be similar to those of the pro-forms of the enzymes isolated from animals that had had their pancreatic duct disconnected 3 days before being killed. These results confirmed earlier findings of the existence of pro-forms of some of the microvillar enzymes and thus indicate a low activity of pancreatic proteinases in the culture system.

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Tyrosine sulphation is not required for microvillar expression of intestinal aminopeptidase N.

Project description:The effect of 2,6-dichloro-4-nitrophenol (DCNP), an inhibitor of phenol sulphotransferases (EC 2.8.2.-), on the biosynthesis of aminopeptidase N (EC 3.4.11.2) was studied in organ-cultured pig intestinal mucosal explants. At 50 microM DCNP did not affect protein synthesis but it decreased incorporation of [35S]sulphate into aminopeptidase N and other major microvillar hydrolases by 70-85% compared with controls, indicating an inhibition of their post-translational tyrosine sulphation. In labelling experiments with [35S]methionine from 0.5 to 5 h, DCNP was tested for its possible influence on synthesis, processing and microvillar expression of aminopeptidase N, but no effect on any of these parameters could be detected. It can therefore be concluded that tyrosine sulphation is not required (for instance as a sorting signal) for the targeting of newly synthesized enzymes to the microvillar membrane.

| S-EPMC1135059 | biostudies-other

Ookinete-interacting proteins on the microvillar surface are partitioned into detergent resistant membranes of Anopheles gambiae midguts.

Project description:Lipid raft microdomains, a component of detergent resistant membranes (DRMs), are routinely exploited by pathogens during host-cell entry. Multiple membrane-surface proteins mediate Plasmodium ookinete invasion of the Anopheles midgut, a critical step in the parasite life cycle that is successfully targeted by transmission-blocking vaccines (TBV). Given that lipid rafts are a common feature of host-pathogen interactions, we hypothesized that they promote the partitioning of midgut surface proteins and thus facilitate ookinete invasion. In support of this hypothesis, we found that five of the characterized Anopheles TBV candidates, including the leading Anopheles TBV candidate, AgAPN1, are present in Anopheles gambiae DRMs. Therefore, to extend the repertoire of putative midgut ligands that can be targeted by TBVs, we analyzed midgut DRMs by tandem mass spectrometry. We identified 1452 proteins including several markers of DRMs. Since glycosylphosphotidyl inositol (GPI)-anchored proteins partition to DRMs, we characterized the GPI subproteome of An. gambiae midgut brush-border microvilli and found that 96.9% of the proteins identified in the GPI-anchored fractions were also present in DRMs. Our study vastly expands the number of candidate malarial TBV targets for subsequent analysis by the broader community and provides an inferred role for midgut plasmalemma microdomains in ookinete cell invasion.

| S-EPMC3208356 | biostudies-literature

Proteins of the kidney microvillar membrane. Enzymic and molecular properties of aminopeptidase W.

Project description:Aminopeptidase W is a newly discovered enzyme of the renal and intestinal brush borders, having been first isolated as a 130 kDa glycoprotein recognized by a monoclonal antibody [Gee & Kenny (1985) Biochem. J. 230, 753-764]. It is particularly effective in the hydrolysis of dipeptides, Glu-Trp (Km 0.57 mM; kcat. 6770 min-1) being a favoured substrate. Dipeptides with tryptophan, phenylalanine or tyrosine in the P1 position were rapidly hydrolysed, but the requirements in respect of the P1 residue were not stringent. The activity of aminopeptidase W is markedly influenced by ionic conditions. The highest activity was observed in 100 mM-Tris/HCl, pH 8; phosphate ions were strongly inhibitory. Activity was also greatly affected by bivalent metal ions, and the magnitude and direction of the effects depended on the nature of the buffer anions and on pH. The most effective inhibitors were amastatin and bestatin. Some thiols also inhibited, but other chelating agents, EDTA and 1,10-phenanthroline, had no effect over the concentration range 1-10 mM. Other group-specific inhibitors, for cysteine, serine or aspartic peptidases, were also ineffective. Some molecular properties were studied. Deglycosylation by treatment with N-glycanase diminished the apparent subunit Mr from 130,000 to 90,000. The enzyme contained zinc, 1.2 atoms/subunit, and in spite of the atypical properties of this enzyme in respect of chelating agents, a zinc-catalysed mechanism is the most probable. Its roles in digestion and in renal function are not yet clear.

| S-EPMC1148244 | biostudies-other

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