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

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.     

Biosynthesis of intestinal microvillar proteins. Dimerization of aminopeptidase N and lactase-phlorizin hydrolase

The pig intestinal brush border enzymes aminopeptidase N (EC 3.4.11.2) and lactase-phlorizin hydrolase (EC 3.2.1.23-62) are present in the microvillar membrane as homodimers. Dimethyl adipimidate was used to cross-link the two [35S]methionine-labeled brush border enzymes from cultured mucosal explants. For aminopeptidase N, dimerization did not begin until 5-10 min after synthesis, and maximal dimerization by cross-linking of the transient form of the enzyme required 1 h, whereas the mature form of aminopeptidase N cross-linked with unchanged efficiency from 45 min to 3 h of labeling. Formation of dimers of this enzyme therefore occurs prior to the Golgi-associated processing, and the slow rate of dimerization may be the rate-limiting step in the transport from the endoplasmic reticulum to the Golgi complex. For lactase-phlorizin hydrolase, the posttranslational processing includes a proteolytic cleavage of its high molecular weight precursor. Since only the mature form and not the precursor of this enzyme could be cross-linked, formation of tightly associated dimers only takes place after transport out of the endoplasmic reticulum. Dimerization of the two brush border enzymes therefore seems to occur in different organelles of the enterocyte.     

Biosynthesis of intestinal microvillar proteins. Forskolin reduces surface expression of aminopeptidase N

The effect of forskolin on the biosynthesis and intracellular transport of pig intestinal aminopeptidase N (EC 3.4.11.2) was studied in organ cultured mucosal explants. The drug which activates adenylate cyclase and hence the cAMP-dependent glycogenolytic pathway did not affect the explant content nor microvillar enrichment of the enzyme. Forskolin, however, caused a decrease in the microvillar expression of aminopeptidase N which developed in a time-dependent manner from about 40% by 80 min to 80% by 4 h of labeling. The intracellular pool size of the transient, high mannose glycosylated form of aminopeptidase N was unaffected by forskolin, indicating a normal synthesis in the rough endoplasmic reticulum. The decrease in surface expression is therefore caused by an induced posttranslational degradation of the enzyme, most likely taking place in the Golgi complex. The degradatory effect on newly synthesized aminopeptidase N was not accompanied by any morphological alterations of the enterocyte; in particular, the microvillar membrane appeared entirely unaffected by forskolin. The results obtained provide evidence for the existence of a posttranslational mechanism, whereby a polarized cell is capable of regulating its expression of apical proteins.     

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

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.     

Biosynthesis of intestinal microvillar proteins. Effect of castanospermine on cell-free synthesis of aminopeptidase N

Pig small intestinal mRNA was translated in a rabbit reticulocyte lysate system supplemented with microsomal membranes. Castanospermine, an inhibitor of glucosidase I, induced a high mannose-glycosylated form of microvillar aminopeptidase N (EC 3.4.11.2) of increased molecular mass, indicating the blocked removal of glucose residues. In contrast to its reduced expression in a mucosal explant system [(1986) Biochem. J. 240, 777-782], this molecular form of aminopeptidase N was at least as abundant in cell-free translation as its normal high mannose-glycosylated counterpart, ruling out degradation taking place in the rough endoplasmic reticulum. Degradation of newly produced, malprocessed enzyme must therefore occur at a later stage during intracellular transport, presumably in the sarcoplasmic reticulum or in transitional elements between this organelle and the Golgi complex.     

Biosynthesis of intestinal microvillar proteins. Expression of aminopeptidase N along the crypt-villus axis

The expression of pig small-intestinal aminopeptidase N (EC 3.4.11.2) along the crypt-villus axis was studied in tangential sections of [35S]-methionine-labelled, organ-cultured explants. The only detectable molecular forms of aminopeptidase N along the crypt-villus axis were polypeptides of Mr 140 000 and 166 000, representing the enzyme in a transient and mature form respectively. The synthesis was at a very low level in the crypt region in experiments with labelling periods ranging from 10 min to 3 h. These findings indicate that crypt cells are not fully committed to the expression of aminopeptidase N, either in its mature or in any other immunoreactive molecular form. The expression of aminopeptidase N was markedly stimulated by dexamethasone (1 microgram/ml). During labelling periods of 3 h, dexamethasone caused an approximately threefold increase in the expression of the enzyme in the crypt cells and a moderate increase of about 20% in the villus cells. Whereas the latter can possibly be ascribed to a general protective effect of dexamethasone on villus architecture, these experiments indicate that crypt cells of mucosa from adult individuals exhibit the same sensitivity to glucocorticoids as does the intestinal epithelium during the prenatal and early postnatal phase.     

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

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.     

Pig intestinal microvillar maltase-glucoamylase. Structure and membrane insertion

The NH2-terminal sequence (25 residues) of amphiphilic single polypeptide chain maltase-glucoamylase (EC 3.2.1.20) was determined by gas-phase sequencing. The result indicates that the NH2-terminal segment anchors the enzyme to the microvillar membrane. The single-chain form and the proteolytically processed two-chain form have two distinct active sites differing in heat stability. However, both sites are sensitive to chonduritol B-epoxide and have similar substrate specificity. The amphiphilic single-chain maltase-glucoamylase and the amphiphilic proteolytically processed form were inserted into liposomes and studied by electron microscopy. The results showed that the enzyme is predominantly present as a homodimeric complex in the membrane.     

Biosynthesis of intestinal microvillar proteins. Surface expression of aminopeptidase N is not affected by chloroquine

The effect of chloroquine on the biosynthesis of pig intestinal aminopeptidase N (EC 3.4.11.2) was studied by labelling with [35S]methionine in organ cultured mucosal explants. The lysosomotropic agent did not alter the molecular size of either the transient or the mature form of the enzyme and did not markedly influence the relative intracellular distribution of the two forms. The microvillar expression of aminopeptidase N during labelling periods of 80-120 min was found to be unaffected by chloroquine. Together these data indicate that pH neutralization of the acidic compartments of the cell bears no consequence on the intracellular transport of the newly synthesized microvillar enzyme. This suggests that the acidic compartments are not involved in the post-Golgi transport and that this, in turn, probably occurs via a constitutive rather than a regulated pathway.     

Proteins of the kidney microvillar membrane. The amphipathic form of dipeptidyl peptidase IV.

Dipeptidyl peptidase IV was solubilized from the microvillar membrane of pig kidney by Triton X-100. The purified enzyme was homogeneous on polyacrylamide-gel electrophoresis and ultracentrifugation, although immunoelectrophoresis indicated that amino-peptidase M was a minor contaminant. A comparison of the detergent-solubilized and proteinase (autolysis)-solubilized forms of the enzyme was undertaken to elucidate the structure and function of the hydrophobic domain that serves to anchor the protein to the membrane. No differences in catalytic properties, nor in sensitivity to inhibition by di-isopropyl phosphorofluoridate were found. On the other hand, several structural differences could be demonstrated. Both forms were about 130,000 subunit mol.wt., but the detergent form appeared to be larger by no more than about 4,000. Electron microscopy showed both forms to be dimers, and gel filtration revealed a difference in the dimeric mol.wt. of about 38 000, mainly attributable to detergent molecules bound to the hydrophobic domain. Papain converted the detergent form into a hydrophilic form that could not be distinguished in properties from the autolysis form. A hydrophobic peptide of about 3500 mol.wt. was identified as a product of papain treatment. The detergent and proteinase forms differed in primary structure. Partial N-terminal amino acid sequences were shown to be different, and the pattern of release of amino acids from the C-terminus by carboxypeptidase Y was essentially similar. The results are consistent with a model in which the protein is anchored to the microvillar membrane by a small hydrophobic domain located within the N-terminal amino acid sequence of the polypeptide chain. The significance of these results in relation to biosynthesis of the enzyme and assembly in the membrane is discussed.     

Biosynthesis of intestinal microvillar proteins. Intracellular processing of lactase-phlorizin hydrolase

The biosynthesis of pig small intestinal lactase-phlorizin hydrolase (EC 3.2.1.23-62) was studied by labelling of organ cultured mucosal explants with [35S]methionine. The earliest detactable form of the enzyme was an intracellular, membrane-bound polypeptide of Mr 225 000, sensitive to endo H as judged by its increased electrophoretic mobility (Mr 210 000 after treatment). The labelling of this form decreased during a chase of 120 min and instead two polypeptides of Mr 245 000 and 160 000 occurred, which both barely had their electrophoretic mobility changed by treatment with endo H. The Mr 160 000 polypeptide is of the same size as the mature lactase-phlorizin hydrolase and was the only form expressed in the microvillar membrane. Together, these data are indicative of an intracellular proteolytic cleavage during transport. The presence of leupeptin during labelling prevented the appearance of the Mr 160 000 form but not that of the Mr 245 000 polypeptide, suggesting that the proteolytic cleavage takes place after trimming and complex glycosylation. The proteolytic cleavage was not essential for the transport since the precursor was expressed in the microvillar membrane in the presence of leupeptin.     

Neutral aminopeptidase and dipeptidyl peptidase IV in the development of collagen II-induced arthritis

This study evaluated the hypothesis that neutral (APN) and dipeptidyl-IV (DPPIV) aminopeptidase activity levels would be critical for the susceptibility to arthritis in collagen-induced model (CIA). The macroscopic signs of arthritis in CIA rats were checked and peripheral blood, synovial fluid and synovial tissue from knee joint were withdrawn. Soluble (SF) and solubilized membrane-bound (MF) fractions from the synovial tissue and peripheral blood mononuclear cells (PBMCs) were obtained. APN and DPPIV activities were fluorometrically quantified. Severe swelling in both the entire hind paws was the minimum criterion to select CIA rats with arthritis. These arthritic rats had high APN in plasma, synovial fluid and SF of the synovial tissue, together with low APN and DPPIV in MF of PBMCs and hallmark histological changes in tibio-tarsal joint. CIA rats with no macroscopic signs of arthritis were diagnosed as resistant and they had low APN in MF of the synovial tissue, low DPPIV in SF of PBMCs and high DPPIV in plasma together with histological aspects of tibio-tarsal joint similar to healthy control rats. Data suggested that APN and DPPIV activity levels are related to the development of arthritis, being protective or inducer of the susceptibility. Understanding what is controlling the compartment-specific changes of these peptidases and looking at ways in which to manipulate their activities may lead to a better knowledge of the arthritic processes and novel treatments.     

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

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.     

Biosynthesis and degradation of altered immature forms of intestinal dipeptidyl peptidase IV in a rat strain lacking the enzyme.

We have used a strain of rat (Fischer 344) lacking brush border membrane dipeptidyl peptidase IV activity to examine its effect on the intestinal assimilation of prolyl peptides. In addition, we have examined the biochemical basis for the enzyme deficiency. An analysis of several brush border membrane hydrolases in different regions of the small intestine demonstrates that these rats lack only dipeptidyl peptidase IV. They also have a greatly reduced ability to hydrolyze and absorb in vivo peptides of the NH2-X-Pro-Y type which are known substrates for the enzyme. Immunoblot analysis with polyclonal and monoclonal antibody indicates that the animals lack an identifiable dipeptidyl peptidase IV protein in intestinal epithelial cells. Levels and types of dipeptidyl peptidase IV mRNA were analyzed in several tissues and found to be similar to that of control animals. Biosynthetic labeling of intestinal explants revealed that two distinct forms (102 and 108 kDa) of dipeptidyl peptidase IV are initially synthesized by deficient rats, in contrast to the single protein (106 kDa) observed in normal animals. Pulse-chase labeling experiments (+/- endoglycosidase H) show that these two altered forms of dipeptidyl peptidase IV, although initially glycosylated with N-linked high mannose carbohydrate, fail to be processed to the mature complex glycosylated form and undergo intracellular degradation.     

Preparative purification of dipeptidyl peptidase IV.

Dipeptidyl-Peptidase IV was purified from pig kidney by ammonium sulfate fractionation, gel filtration, QAE-cellulose chromatography and affinity columns with Gly-Pro- and Concanavalin A-Sepharose. The specific activity of the purified enzyme is 41.8 units/mg. Polyacrylamide gel electrophoresis and silver staining show a single band. The enzyme preparation is free of aminopeptidase and dipeptidase activity, proved fluorimetrically and by gas chromatography/mass spectrometry. The most important procedure for removal of contaminating enzyme activities is a stepwise NaCl-gradient on a QAE-ZetaPrep ion exchange disk.     

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

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.     

Interaction of purified brush-border membrane aminopeptidase N and dipeptidyl peptidase IV with lectin-sepharose derivatives

The glycoprotein nature of two peptidases purified from the rat intestinal brush-border membrane was examined by their interaction with several lectin-Sepharose derivatives. Aminopeptidase N (EC 3.4.11.2), which contains 20% carbohydrate by weight, was bound minimally (less than 30%) by columns of Con A-, RCAI- and WGA-Sepharose. Alternatively, a greater proportion of dipeptidyl peptidase IV (EC 3.4.14.-) was bound by these immobilized lectins with 50% of the enzyme binding to Con A-Sepharose. Treatment of both enzymes with neuraminidase enhanced the binding of aminopeptidase to RCAI-Sepharose by 4-fold but did not alter the binding patterns of dipeptidyl peptidase IV. A sequential fractionation of the two peptidases with columns of Con A- and RCAI-Sepharose gave four fractions of each enzyme with differing lectin-binding specificities. Approximately 60% of the dipeptidyl peptidase IV interacted with either one or both of the lectins while only 30% of the aminopeptidase N did so. Kinetic analysis of the four isolated fractions revealed some differences, possibly related to variations in the carbohydrate moiety. The findings confirm that these two purified rat intestinal brush-border membrane peptidases are glycoproteins and, while they share a common physiologic function and source, they apparently have very different and possibly unique asparagine-linked oligosaccharide side-chains. In addition, a considerable degree of microheterogeneity exists in the carbohydrate structure of these two enzymes.     

Biosynthesis of intestinal microvillar proteins. Low temperature arrests both processing and intracellular transport

The effect of culture at 20 degrees C on biosynthesis of microvillar enzymes was studied in pig small intestinal mucosal explants. At this temperature, aminopeptidase N (EC 3.4.11.2) and sucrase-isomaltase (EC 3.2.1.48-10) both accumulated intracellularly, predominantly in their transient, high mannose-glycosylated form characteristic of the newly synthesized enzymes prior to the molecular processing taking place in the Golgi complex. The general morphology of the enterocyte was unaffected by culture at low temperature except for the Golgi complex where the cisternae appeared condensed and surrounded by numerous vesicles of 50 to 55 nm. Both molecular processing and microvillar expression could be restored by shifting the temperature to 37 degrees C. Culture at low temperature did not induce any missorting of newly synthesized aminopeptidase N, but both molecular processing and microvillar expression only resumed at a slow rate after increasing the temperature, suggesting that reorganization of the Golgi complex is a time-requiring process.     

Purification and molecular characterization of rat intestinal brush border membrane dipeptidyl aminopeptidase IV

Dipeptidyl aminopeptidase IV (EC 3.4.14.-) was solubilized from a particulate membrane fraction of rat intestinal mucosa with Triton X-100. The solubilized enzyme was purified to homogeneity following ammonium sulfate fractionation, chromatography on DEAE-Sepharose and hydroxyapatite, gel filtration and preparative polyacrylamide gel electrophoresis. The final enzyme preparation had a specific activity of 55 units/mg protein representing a 1373 fold purification over the starting material. Purity was judged by polyacrylamide gel electrophoresis and double immunodiffusion. The molecular weight of the native undenatured enzyme was estimated to be 230000 by gel filtration and polyacrylamide gel electrophoresis. Electrophoresis under denaturing conditions (sodium dodecyl sulfate) indicated that the protein consists of two identical 98 kDa subunits. Dipeptidyl aminopeptidase IV is a glycoprotein containing approx. 8% carbohydrate by weight. A detailed analysis of the individual sugar components demonstrated that fucose, galactose, glucose, mannose, sialic acid and hexosamine sugars were present. The nature of the constituent asparagine linked oligosaccharide side chains was further examined following cleavage from the peptide backbone by hydrazinolysis. Following high voltage paper electrophoresis approx. 80% of the isolated oligosaccharide was found with the neutral fraction while the remaining 20% consisted of a single acidic component. Gel filtration of the neutral oligosaccharide fraction indicated that it contains approx. 19 sugar residues.     

Stability and bioactivity studies on dipeptidyl peptidase IV resistant glucogan-like peptide-1 analogues

Glucagon-like peptide-1 (GLP-1) was once considered as an ideal anti-diabetic candidate for its important role in maintaining glucose homeostasis through the regulation of islet hormone secretion, as well as hepatic and gastric function. However, the major therapeutic obstacle for using native GLP-1 as a therapeutic agent is its very short half-life primarily due to their degradation by the enzyme dipeptidyl peptidase IV (DPP-IV). In this study, GLP-1 analogues with modifications in amino acid site 8, 22 and 23 were synthesized using solid phase peptide synthesis. Resistance of these analogues to DPP-IV cleavage was investigated in vitro by incubation of the peptides with DPP-IV or human plasma. Glucoregulating efficacy of the analogues was evaluated in normal Kunming mice using intraperitoneal glucose tolerance model. Glucose lowering effect of combination therapy (analogue plus Vildagliptin) has also been studied. In vitro studies showed that the modified analogues were much more stable than native GLP-1 (nearly 100% of the peptide keep intact after 4 h incubation). In vivo biological activity evaluation revealed that His8-EEE (the most potent GLP-1 analogues in this study) exhibited significantly improved glycemic control potency (approximately 4.1-fold over saline and 2.5-fold over GLP-1) and longer time of active duration (at least 5 h). Combination therapy also showed the trend of its superiority over mono-therapy. Modified analogues showed increased potency and biological half-time compared with the native GLP-1, which may help to understand the structure-activity relationship of GLP-1 analogues.