Hanne Skovbjerg

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.

Further characterization of intestinal lactase/phlorizin hydrolase

Pig intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purified enzyme was free of other known brush border enzymes and appeared homogeneous in immunoelectrophoresis and polyacrylamide gel electrophoresis in the presence of SDS. Pig lactase/phlorizin hydrolase was shown to have the same quaternary structure as the human enzyme, i.e., built up of two polypeptides of the same molecular weight (160000). In addition to hydrolyzing lactose, phlorizin and a number of synthetic substrates, both the human and the pig enzyme were shown to have a considerable activity against cellotriose and cellotetraose, and a low but significant activity against cellulose. The lactase/phlorizin hydrolase isolated from pigs in which the pancreatic ducts had been disconnected 3 days before death and from Ca2+-precipitated enterocyte membranes (basolateral and intracellular membranes) exhibited in SDS-polyacrylamide gel electrophoresis the same size of constituent polypeptides and the same catalytic and immunological properties as a normal brush border lactase/phlorizin hydrolase.

Immunoelectrophoretic studies on human small intestinal brush border proteins—the longitudinal distribution of peptidases and disaccharidases

The longitudinal distribution of different brush border enzymes along the human small intestine was studied by crossed immunoelectrophoresis. The results are based on biopsies taken every 50 cm in three intestines obtained at autopsy and on peroral or peroperative biopsies from the ligament of Treitz, proximal jejunum and distal ileum from 11 patients undergoing jejunoileal bypass operation for obesity. Lactase-phlorizin hydrolase (EC 3.2.1.23-62) and sucrase-isomaltase(EC 3.2.1.48-10) had their highest level in jejunum with decreasing activity towards the proximal and distal ends of the intestine, while maltase (EC 3.2.1.20) increased along the intestine and reached its highest activity in the distal ileum. A carboxypeptidase (EC 3.4.12.X) is demonstrated as an enzymatic entity of the human intestine. This enzyme had a rather flat distribution curve while microvillus aminopeptidase (EC 3.4.11.2), dipeptidyl peptidase IV (EC 3.4.14.X) and aspartate aminopeptidase (EC 3.4.11.7) all increased along the length axis and reached maximum values in distal ileum.

Evidence for biosynthesis of lactase-phlorizin hydrolase as a single-chain high-molecular weight precursor

Precursor forms of lactase-phlorizin hydrolase, sucrase-isomaltase and aminopeptidase N were studied by pulse-labelling of organ-cultured human intestinal biopsies. After labelling the biopsies were fractionated by the Ca2+-precipitation method and the enzymes isolated by immunoprecipitation. The results indicate that the lactase-phlorizin hydrolase is synthesized as a Mr 245 000 polypeptide, which is intracellularly cleaved into its mature Mr 160 000 form. Sucrase-isomaltase is shown to be synthesized as a single chain precursor (Mr 245 000 and 265 000) while the precursor of aminopeptidase N is shown to be of apparently the same size as the mature enzyme (Mr 140 000 and 160 000).

Gliadin is a Good Substrate of Several Transglutaminases: Possible Implication in the Pathogenesis of Coeliac Disease

Background: Deamidation of distinct glutamines in HLA-DQ2 restricted gliadin epitopes, considered critical in the pathogenesis of coeliac disease (CD), can be mediated by tissue transglutaminase (tTG). To elucidate the possible role of other transglutaminases in CD we investigated whether different mammalian, microbial and vegetable transglutaminases can use gliadin as substrate. Methods: Studies in which small amounts of transglutaminase have been measured have led to our modifying a microtitre plate assay. We used proteolytically digested gliadin as solid phase substrate and Europium-labelled streptavidine to quantify the biotinylated product covalently linked by the enzyme to the plate. Results: The modified assay is ultrasensitive and quantitative, measuring guinea pig liver transglutaminase concentrations between 0.5 and 50 ng/well. The specific activities of the enzymes (counts/min/mg) against gliadin and N,N-dimethylcasein, respectively, are: tTG 9800/4900, Factor XIII 97330/55620, epidermal transglutaminase 47650/50770, streptoverticillium transglutaminase 4290/2200, phytophora cactorum transglutaminase 6910/4110. For the first time, we have detected transglutaminase activity in bean sprouts, spinach leaves and green peas, which are commonly used vegetables. Conclusion: Gliadin is a good substrate for endogenous, microbial and plant transglutaminases. An interesting alternative is that gliadins are deamidated by microbial or food transglutaminases in the intestinal lumen. The assay described provides an ultrasensitive method for measuring small amounts of transglutaminase and is considered a helpful tool in further studies of the possible role of transglutaminases in the pathogenesis of CD.

Biosynthesis of intestinal microvillar proteins. Nature of precursor forms of microvillar enzymes from Ca2+-precipitated enterocyte membranes.

In the synthesis of membrane glycoproteins, the existence of a common asparagine-linked oligosaccharide intermediate, characterized by possessing a large number of mannose residues (high mannose form) has been proposed [l]. This intermediate is in turn modified by partial reglycosylation, yielding the final complex oligosaccharides, characterized by terminal sialic acid or fucose residues. Antibodies against denatured maltase-glucoamylase were prepared as follows: The pure enzyme [S] was boiled for 5 min in the presence of 1% SDS. Excess of SDS was removed by precipitation with 0.05 M KC1 (final cont.). After mixing with an equal volume of incomplete Freunds adjuvant, the der.atured enzyme was injected into rabbits (50 fig/injection) at 2 week intervals. A week after the fourth injection, the rabbits were bled for 40 ml.

Immunoelectrophoretic studies on human small intestinal brush border proteins A qualitative study of the protein composition

The human small intestinal brush border proteins were studied qualitatively by crossed immunoelectrophoresis. Brush border membranes were purified from human jejunum and the proteins released by Triton X-100. Rabbits were immunized with the released proteins and by using a double layer immunofluorescence technique the obtained antisera were shown to be specific against the brush border proteins. The precipitates obtained in crossed immunoelectrophoresis were identified by enzymatic staining techniques. Sucrase (EC 3.2.1.48), isomaltase EC 3.2.1.10), maltase (EC 3.2.1.20), phloretin-glucosidase (EC 3.2.1.62), lactase (EC3.2.1.23), microvillus aminopeptidase (aminopeptidase (microsomal), EC 3.4.11.2), dipeptidyl peptidase IV (EC 3.4.14.X), and alkaline phosphatase (EC 3.1.3.1) were identified while asparate aminopeptidase (EC 3.4.11.7), gamma-glutamyl transferase (EC 2.3.2.2) and trehalase (EC 3.2.1.28) could not be visualized. This work demonstrates that cross immunoelectrophoresis can be used in the study of human small intestinal brush border proteins.

Collagen mRNA levels changes during colorectal cancer carcinogenesis

BackgroundInvasive growth of epithelial cancers is a complex multi-step process which involves dissolution of the basement membrane. Type IV collagen is a major component in most basement membranes. Type VII collagen is related to anchoring fibrils and is found primarily in the basement membrane zone of stratified epithelia. Immunohistochemical studies have previously reported changes in steady-state levels of different α(IV) chains in several epithelial cancer types. In the present study we aimed to quantitatively determine the mRNA levels of type IV collagen (α1/α4/α6) and type VII collagen (α1) during colorectal cancer carcinogenesis.MethodsUsing quantitative RT-PCR, we have determined the mRNA levels for α1(IV), α4(IV), α6(IV), and α1(VII) in colorectal cancer tissue (n = 33), adenomas (n = 29) and in normal tissue from the same individuals. In addition, corresponding tissue was examined from healthy volunteers (n = 20). mRNA levels were normalized to β-actin. Immunohistochemical analysis of the distributions of type IV and type VII collagens were performed on normal and affected tissues from colorectal cancer patients.ResultsThe α1(IV) and α1(VII) mRNA levels were statistically significantly higher in colorectal cancer tissue (p < 0.001) as compared to corresponding tissue from healthy controls. This is an early event as tissue from adenomas also displayed a higher level. There were small changes in the levels of α4(IV). The level of α6(IV) was 5-fold lower in colorectal cancer tissue as compared to healthy individuals (p < 0.01). The localisation of type IV and type VII collagen was visualized by immunohistochemical staining.ConclusionOur results suggest that the down-regulation of α6(IV) mRNA coincides with the acquisition of invasive growth properties, whereas α1(IV) and α1(VII) mRNAs were up-regulated already in dysplastic tissue. There are no differences in collagen expression between tissues from healthy individuals and normal tissues from affected individuals.

Hepatocyte growth factor activator inhibitor-2 prevents shedding of matriptase.

Hepatocyte growth factor activator inhibitor-2 (HAI-2) is an inhibitor of many proteases in vitro, including the membrane-bound serine protease, matriptase. Studies of knock-out mice have shown that HAI-2 is essential for placental development only in mice expressing matriptase, suggesting that HAI-2 is important for regulation of matriptase. Previous studies have shown that recombinant expression of matriptase was unsuccessful unless co-expressed with another HAI, HAI-1. In the present study we show that when human matriptase is recombinantly expressed alone in the canine cell line MDCK, then human matriptase mRNA can be detected and the human matriptase ectodomain is shed to the media, suggesting that matriptase expressed alone is rapidly transported through the secretory pathway and shed. Whereas matriptase expressed together with HAI-1 or HAI-2 accumulates on the plasma membrane where it is activated, as judged by cleavage at Arg614 and increased peptidolytic activity of the cell extracts. Mutagenesis of Kunitz domain 1 but not Kunitz domain 2 abolished this function of HAI-2. HAI-2 seems to carry out its function intracellularly as this is where the vast majority of HAI-2 is located and since HAI-2 could not be detected on the basolateral plasma membrane where matriptase resides. However, minor amounts of HAI-2 not undergoing endocytosis could be detected on the apical plasma membrane. Our results suggest that Kunitz domain 1 of HAI-2 cause matriptase to accumulate in a membrane-bound form on the basolateral plasma membrane.

Deamidation of Gliadin Peptides in Lamina Propria : Implications for Celiac Disease

Activation of small intestinal gluten-reactive CD4+ T-cells is a critical event in celiac disease. Deamidation of specific glutamine residues by tissue transglutaminase enhances the binding of T-cell activating gliadin epitopes to DQ2, increasing T-cell recognition. Our purpose was to investigate whether deamidated gliadin epitopes can be generated in the small intestinal mucosa by tissue transglutaminase and to characterize the location of the process. Intestinal explants from pig intestine and frozen biopsy slices from human and rat intestine were incubated with α-gliadin peptides containing the immunodominant motif. Monoclonal antibodies specifically recognizing the non-deamidated and/or the deamidated epitope were used for immunofluorescence studies. We conclude that endogenous tissue transglutaminase can mediate extracellular deamidation of gliadin peptides in the lamina propria. Gliadin peptides with more than one recognition site can be simultaneously cross-linked and deamidated extracellularly in the lamina propria, and might be of importance for the antibody response seen in untreated celiac disease patients.