Tight and specific lanthanide binding in a de novo TIM barrel with a large internal cavity designed by symmetric domain fusion.

De novo protein design has succeeded in generating a large variety of globular proteins, but the construction of protein scaffolds with cavities that could accommodate large signaling molecules, cofactors, and substrates remains an outstanding challenge. The long, often flexible loops that form such cavities in many natural proteins are difficult to precisely program and thus challenging for computational protein design. Here we describe an alternative approach to this problem. We fused two stable proteins with C2 symmetry-a de novo designed dimeric ferredoxin fold and a de novo designed TIM barrel-such that their symmetry axes are aligned to create scaffolds with large cavities that can serve as binding pockets or enzymatic reaction chambers. The crystal structures of two such designs confirm the presence of a 420 cubic Ångström chamber defined by the top of the designed TIM barrel and the bottom of the ferredoxin dimer. We functionalized the scaffold by installing a metal-binding site consisting of four glutamate residues close to the symmetry axis. The protein binds lanthanide ions with very high affinity as demonstrated by tryptophan-enhanced terbium luminescence. This approach can be extended to other metals and cofactors, making this scaffold a modular platform for the design of binding proteins and biocatalysts.

Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease.

The action of tissue Transglutaminase (TGase) on specific protein-bound glutamine residues plays a critical role in numerous biological processes. Here we provide evidence for a new role of this enzyme in the common, HLA-DQ2 (and DQ8) associated enteropathy, celiac disease (CD). The intestinal inflammation in CD is precipitated by exposure to wheat gliadin in the diet and is associated with increased mucosal activity of TGase. This enzyme has also been identified as the main target for CD-associated anti-endomysium autoantibodies, and is known to accept gliadin as one of its few substrates. We have examined the possibility that TGase could be involved in modulating the reactivity of gliadin specific T cells. This could establish a link between previous reports of the role of TGase in CD and the prevailing view of CD as a T-cell mediated disorder. We found a specific effect of TGase on T-cell recognition of gliadin. This effect was limited to gliadin-specific T cells isolated from intestinal CD lesions. We demonstrate that TGase mediates its effect through an ordered and specific deamidation of gliadins. This deamidation creates an epitope that binds efficiently to DQ2 and is recognized by gut-derived T cells. Generation of epitopes by enzymatic modification is a new mechanism that may be relevant for breaking of tolerance and initiation of autoimmune disease.

Colonic Health in Hospitalized Horses Treated with Non-Steroidal Anti-Inflammatory Drugs - A Preliminary Study.

Non-steroidal anti-inflammatory drugs (NSAIDs) can cause right dorsal colitis, but longitudinal clinical studies are lacking. This study investigates whether NSAID treated horses develop right dorsal colonic pathology in a clinical setting. Non-gastrointestinal hospitalized horses treated with NSAIDs >4 days, and untreated hospital-owned teaching horses and non-gastrointestinal client-owned hospitalized horses were included. All horses were monitored over time with clinical examinations (focusing on presence of colic, depression, reduced appetite, unstructured feces), ultrasonographic intestinal wall measurements, fecal occult blood tests (semi-quantitative results), and blood analysis (total protein and albumin concentrations, white blood cell and neutrophil counts). Outcomes were recorded as "ultrasonographically thickened right dorsal colon (RDC) walls", "colitis" and "right dorsal colitis". Findings over time were compared to baseline values and to control horses. Seventeen NSAID treated horses and 5 controls were included. NSAID treated horses developed thickened RDC walls (4/9), and subclinical and mild colitis (9/11) and right dorsal colitis (4/10), whereas all control horses remained healthy. The first changes were identified on treatment day 2. RDC walls of treated horses were significantly thicker compared to their own baseline values and compared to control horses. In conclusion, presumptive colon pathology was identified with a high incidence, starting early in the course of treatment, but with low severity. Appropriate monitoring should be advised throughout NSAID treatment. Additional research for noninvasive diagnostic tests for colon pathology is required.

Aminopeptidase N is directly sorted to the apical domain in MDCK cells.

In different epithelial cell types, integral membrane proteins appear to follow different sorting pathways to the apical surface. In hepatocytes, several apical proteins were shown to be transported there indirectly via the basolateral membrane, whereas in MDCK cells a direct sorting pathway from the trans-Golgi-network to the apical membrane has been demonstrated. However, different proteins had been studied in these cells. To compare the sorting of a single protein in both systems, we have expressed aminopeptidase N, which already had been shown to be sorted indirectly in hepatocytes, in transfected MDCK cells. As expected, it was predominantly localized to the apical domain of the plasma membrane. By monitoring the appearance of newly synthesized aminopeptidase N at the apical and basolateral surface, it was found to be directly sorted to the apical domain in MDCK cells, indicating that the sorting pathways are indeed cell type-specific.

The occurrence of aminopeptidase N and desquamated cell proteins in intestinal perfusate of the rat.

The in vivo release of a microvillar enzyme, aminopeptidase N (EC 3.4.11.2), and a cytosolic enzyme, lactate dehydrogenase (EC 1.1.1.27), into the intestinal lumen was measured to gain information on the fraction of desquamated cell protein in intestinal juice and on the mechanism of release of intestinal microvillar enzymes. 1.6% and 2.9% of the mucosal activities of aminopeptidase N and lactate dehydrogenase were released to the intestinal lumen per hour, respectively. The ratios between aminopeptidase N and lactate dehydrogenase in intestinal perfusates and mucosal homogenates were similar. This result is compatible with the view that aminopeptidase N in the rat small intestine is predominantly released into the intestinal lumen by desquamation of enterocytes. This conclusion was supported by the failure to demonstrate microvesiculation of the microvilli by electron microscopy. 30-40% of the aminopeptidase N in the intestinal lumen is membrane-bound indicating that partial solubilization occurs during desquamation. The addition of calcium ions did not augment the release of aminopeptidase N or the membrane-bound fraction in the lumen. 10-20% of the protein content in the intestinal lumen is due to extruded cell protein. This includes aminopeptidase N which constitutes 1% of the luminal protein.

Immunoelectrophoretic studies on pig intestinal brush border proteins.

Brush borders were prepared from pig intestinal mucosa and the membrane proteins solubilized with either Triton X-100 or papain. Proteins, thus released, were used as antigens to raise antisera in rabbits. The immunoglobulin G fractions were isolated and shown by the double layer immunofluorescence staining technique to react only with the brush border region of the enterocyte. The antibodies obtained were used in immunoelectrophoretic studies on the brush border proteins. Eight hydrolytic activities were identified by the use of histo-chemical staining methods. These were the microsomal aminopeptidase (EC 3.4.11.2), aspartate aminopeptidase (EC 3.4.11.7), dipeptidyl peptidase IV (EC 3.4.14.X), lactase (EC 3.2.1.23), glucoamylase (EC 3.2.1.3), sucrase (EC 3.2.1.48), isomaltase (EC 3.2.1.10) and alkaline phosphatase (EC 3.1.3.1). In addition, at least four faint immunoprecipitates were formed but none of these were identified.

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.

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).

Immunomicroscopic localization of aminopeptidase N in the pig enterocyte. Implications for the route of intracellular transport.

The subcellular localization of aminopeptidase N (EC 3.4.11.2) in the pig enterocyte was investigated by immunofluorescence and immunoelectron microscopy (immunogold staining). By indirect immunofluorescence on either frozen or paraffin-embedded sections, a very intense staining in the microvillar membrane and a weak intracellular staining was demonstrated. No staining was detected in the basolateral membrane. Likewise, the immunogold labelling on Epon-embedded sections was concentrated in the microvillar membrane, whereas the basolateral membrane did not contain significant amounts of labelling. Labelling was demonstrated in the Golgi apparatus and in a minor fraction of the intracellular smooth vesicles positioned between the Golgi apparatus and the microvillar membrane. These observations are compatible with the view that newly synthesized aminopeptidase N is delivered directly to the microvillar membrane by smooth vesicles having a diameter about 70 to 100 nm and does not pass the basolateral membrane on its way to the brush border membrane.

Organelles involved in the intracellular transport of newly synthesized aminopeptidase N and their acidity.

The intracellular routes taken by aminopeptidase N, an apically expressed enzyme in the enterocyte, was followed in small intestinal cultures of pig using either immunoelectron microscopy (immunogold labeling) or continuous labeling with [35S]methionine. Aminopeptidase N was found in the microvillar membrane, the Golgi complex, apical small smooth vesicles, and various acidic lysosomal/endosomal-like organelles. By culturing mucosal explants in the presence of either cycloheximide or (3-(2,4-dinitroanilino)-3-amino-N-methylpropylamine) (DAMP) it was demonstrated that the apical small smooth vesicles are exocytotic and that the low pH in the acid compartments is of no importance for intracellular transport and correct sorting of aminopeptidase N. Furthermore, our results show that the majority of the aminopeptidase N in the lysosomal/endosomal-like compartments is newly synthesized.

Cloning of the pig aminopeptidase N gene. Identification of possible regulatory elements and the exon distribution in relation to the membrane-spanning region.

We have isolated four lambda-phages covering the complete pig aminopeptidase N/CD13 gene. The sequence of 2.85 kbp encompasses 1.18 kbp of the 5' upstream region and 1.67 kbp of the structural gene. In the promoter region we find a TATA box and potential binding sites for CTF-1/NF-1 and AP-2. By sequence comparisons we have found three domains showing similarity to promoter regions of the genes encoding human alpha 1-antitrypsin and human intestinal alkaline phosphatase. The gene sequence includes the first three exons and two introns. It shows that a single exon encodes the cytoplasmic tail, the membrane anchor and the junctional peptide.

The apical sorting signal on human aminopeptidase N is not located in the stalk but in the catalytic head group.

Human aminopeptidase N carries an apical sorting signal on its ectodomain necessary for its correct transport to the apical membrane in Madin-Darby canine kidney cells. To determine whether the apical sorting signal is localized in the serine/threonine rich stalk or in the catalytic head group, anchor/stalk-minus aminopeptidase N, consisting of the hemagglutinin signal peptide and the catalytic head group of human aminopeptidase N, was expressed in MDCK cells. Anchor/stalk-minus aminopeptidase N was secreted mainly to the apical side. The catalytic head group of human aminopeptidase N thus carries an apical sorting signal.

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.

Uteroglobin, an apically secreted protein of the uterine epithelium, is secreted non-polarized form MDCK cells and mainly basolaterally from Caco-2 cells.

A complete cDNA encoding rabbit uteroglobin was constructed and expressed in MDCK and Caco-2 cells. The MDCK cells secrete uteroglobin in approximately equal amounts to the apical and the basolateral side, whereas the Caco-2 cells secrete uteroglobin mainly to the basolateral side. Both MDCK and Caco-2 cells thus secrete uteroglobin in a non-sorted manner. It has, however, previously been shown that uteroglobin is secreted exclusively at the apical membrane in primary cell culture of endometrial epithelial cells [S.K. Mani et al. (1991) Endocrinology 128, 1563-1573]. This suggests that either the endometrial epithelium has an apical default pathway or recognises a sorting signal not recognised by MDCK cells and Caco-2 cells. Our data thus show that a soluble molecule can be secreted at the apical, the basolateral or both membranes depending on the cell type.

Onset of transcription of the aminopeptidase N (leukemia antigen CD 13) gene at the crypt/villus transition zone during rabbit enterocyte differentiation.

The sequence of a cDNA clone (2.82 kbp) of rabbit intestinal aminopeptidase N (CD 13) is reported. Using the corresponding anti-sense RNA probe, the distribution of aminopeptidase N mRNA along the crypt/villus axis of the rabbit small intestine was studied by in situ hybridization. The aminopeptidase N gene is expressed along the whole length of the villus with a maximum at its base. Expression was not detected in the crypt cells. The distribution of aminopeptidase N mRNA correlates with the presence of active enzyme as monitored by histochemical staining. The results are compatible with onset of transcription of the aminopeptidase N gene at the crypt/villus transition zone during the enterocyte differentiation.

Two intestinal specific nuclear factors binding to the lactase-phlorizin hydrolase and sucrase-isomaltase promoters are functionally related oligomeric molecules.

Lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are enterocyte-specific gene products. The identification of regulatory cis-elements in the promoter of these two genes has enabled us to carry out comparative studies of the corresponding intestinal-specific nuclear factors (NF-LPH1 and SIF1-BP). Electrophoretic mobility shift assays demonstrated that the two nuclear factors compete for binding on the same cis-elements. The molecular size of the DNA binding polypeptide is estimated to be approximately 50 kDa for both factors. In the native form the factors are found as 250 kDa oligomeric complexes. Based on these results NF-LPH1 and SIF1-BP are suggested to be either identical or closely related molecules.

Assignment of the human aminopeptidase N (peptidase E) gene to chromosome 15q13-qter.

The gene for aminopeptidase N (EC 3.4.11.2) has been located on the human chromosome 15q13-qter using HindIII-cleaved DNA from a panel of hybrids between rodent and human cells. The Southern blots were probed by the 5'-EcoRI fragment of the recently cloned human aminopeptidase N cDNA.

The human lactase-phlorizin hydrolase gene is located on chromosome 2.

The lactase-phlorizin hydrolase gene was assigned to chromosome 2 by analysis of Southern blots of DNA from a panel of human-rodent cell hybrids containing characteristic sets of human chromosomes The hybridization probe used was a recently isolated cDNA clone of the human lactase-phlorizin hydrolase gene.

1 kb of the lactase-phlorizin hydrolase promoter directs post-weaning decline and small intestinal-specific expression in transgenic mice.

Adult-type hypolactasia is a genetic condition making approximately one half of the human population intolerant to milk because of abdominal symptoms. The cause is a post-weaning down-regulation of the intestinal-specific enzyme lactase-phlorizin hydrolase (LPH) reducing the intestinal capacity to hydrolyze lactose. We here demonstrate that the stretch -17 to -994 in the pig LPH-promoter carries cis-elements which direct a small intestinal-specific expression and a post-weaning decline of a linked rabbit beta-globin gene. These data demonstrate that the post-weaning decline of LPH is mainly due to a transcriptional down-regulation.

N-Terminal sequences of pig intestinal sucrase-isomaltase and pro-sucrase--isomaltase. Implications for the biosynthesis and membrane insertion of pro-sucrase--isomaltase.

The hog sucrase-isomaltase complex is anchored to the small-intestinal brush border membrane, as in the rabbit, via a hydrophobic segment located in the N-terminal region of the isomaltase subunit. The immediate precursor of the 'final' sucrase-isomaltase (i.e., pro-sucrase-isomaltase as prepared from adult hogs whose pancreas had been disconnected from the duodenum) is an amphiphilic single polypeptide chain of Mr 260000-265000. Its N-terminal sequence is virtually identical with (not merely homologous to) the corresponding region of the isomaltase subunit of 'final' sucrase-isomaltase. This shows that the isomaltase portion of pro-sucrase-isomaltase is the N-terminal 'half' of the precursor polypeptide chain. Thus the succession of domains in pro-sucrase-isomaltase and its mode of anchoring in the membrane could be deduced. On this basis a likely mechanism of biosynthesis and insertion is proposed.