Ove Norén

Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA

The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250–555 positioned within the catalytic domain shows very clear homology to E. coli aminopeptidase N and contains Zn2+ ligands. Therefore these residues are part of the active site. However, no homology of the anchor/junctional peptide domain is found suggesting that the juxta‐ and intra‐membraneous parts of the molecule have been added/preserved during development. It is speculated that this part carries the apical address.

Interaction between the homeodomain proteins Cdx2 and HNF1alpha mediates expression of the lactase-phlorizin hydrolase gene.

Lactase-phlorizin hydrolase is a brush-border enzyme which is specifically expressed in the small intestine where it hydrolyses lactose, the main carbohydrate found in milk. We have previously demonstrated in transgenic mice that the tissue-specific and developmental expression of lactase is controlled by a 1 kb upstream region of the pig lactase gene. Two homeodomain transcription factors, caudal-related homeodomain protein (Cdx2) and hepatic nuclear factor 1alpha (HNF1alpha), are known to bind to regulatory cis elements in the promoters for several intestine-specific genes, including lactase, and are present in mammalian intestinal epithelia from an early stage in development. In the present study, we examined whether Cdx2 and HNF1alpha physically interact and co-operatively activate transcription from the lactase-phlorizin hydrolase promoter. We show that the presence of both factors leads to a much higher level of transcription than the sum of the activation by either factor alone. The N-terminal activation domain of Cdx2 is required for maximal synergy with HNF1alpha. With the use of pull-down assays, we demonstrate a direct protein-protein interaction between Cdx2 and HNF1alpha. The interaction domain includes the homeodomain region of both proteins. This is the first demonstration of a functional interaction between two transcription factors involved in the activation of a number of intestine-specific genes. Synergistic interaction between tissue-restricted factors is likely to be an important mechanism for reinforcing developmental and tissue-specific gene expression within the intestine.

The Prenatal Development and Glucocorticoid Control of Brush-Border Hydrolases in the Pig Small Intestine

The development of brush-border enzymes and the possible regulatory role of cortisol were investigated in the small intestine of the fetal and neonatal pig. With the sows under pentobarbitone anesthesia, osmotic minipumps containing either saline or cortisol were inserted s.c. into 25 fetuses from 10 pregnant sows (82–96 d gestation). Six d later, the infused fetuses were removed by cesarean section and samples of the proximal, middle, and distal intestine taken for analysis. Samples were also obtained from 48 piglets that did not undergo an operation (controls) and that were removed at intervals from 82 d gestation until term (114 ± 2 d). In the proximal and middle intestine, the mean levels of lactase-phlorizin hydrolase (EC 3.2.1.23–62), maltase-glucoamylase (EC 3.2.1.20), aminopeptidase N (EC 3.4.11.2), and aminopeptidase A (EC 3.4.11.7) increased during the last 10–15 d before term, correlated positively with log10 plasma cortisol values, and were higher in cortisol-infused than in saline-infused fetuses (p < 0.05). Activity of sucrase-isomaltase (EC 3.2.1.48–10) was low in fetal pigs, and this enzyme and dipeptidyl peptidase IV (EC 3.4.14.5) were not significantly affected by fetal age or exogenous cortisol. Maltase (EC 3.2.1.48–10 and EC 3.2.1.20) activity was significantly decreased in the middle and distal intestine of cortisol-infused fetuses. The results suggest that the prepartum rise in endogenous cortisol secretion stimulates the prenatal expression of certain brush-border enzymes in the pig small intestine at this critical time. However, the effects of cortisol on the developing intestine were highly idiosyncratic for particular enzymes and intestinal regions.

Further Characterization of Aminopeptidase-N as a Receptor for Coronaviruses

We recently reported that porcine aminopeptidase-N (pAPN) acts as a receptor for transmissible gastroenteritis virus (TGEV). In the present work, we addressed the question of whether TGEV tropism is determined only by the virus-receptor interaction. To this end, different non-permissive cell lines were transfected with the porcine APN cDNA and tested for their susceptibility to TGEV infection. The four transfected cell lines shown to express pAPN at their membrane became sensitive to infection. Two of these cell lines were found to be defective for the production of viral particles. This suggests that other factor(s) than pAPN expression may be involved in the production of infectious virions. The pAPN-transfected cells were also tested for their susceptibility to several viruses which have a close antigenic relationship to TGEV. So far, we failed to evidence permissivity to the feline infectious peritonitis coronavirus FIPV and canine coronavirus CCV. In contrast, we found clear evidence that porcine respiratory coronavirus PRCV, a variant of TGEV which replicates efficiently in the respiratory tract but to a very low extent in the gut, may also utilise APN to gain entry into the host cells. This suggests that the switch between TGEV and PRCV tropisms in vivo may involve other determinant(s) than receptor recognition.

Human Coronavirus 229E: Receptor Binding Domain and Neutralization by Soluble Receptor at 37°C

ABSTRACT Truncated human coronavirus HCoV-229E spike glycoproteins containing amino acids 407 to 547 bound to purified, soluble virus receptor, human aminopeptidase N (hAPN). Soluble hAPN neutralized the infectivity of HCoV-229E virions at 37°C, but not 4°C. Binding of hAPN may therefore trigger conformational changes in the viral spike protein at 37°C that facilitate virus entry.

Purification and specificity of pig intestinal prolidase

Abstract Purification of prolidase (imidodipeptidase, EC 3.4.3.7) from pig small intestine is described. Two alternative purification procedures have been developed, one of which utilizes the capability of the enzyme to aggregate to high molecular weight complexes and allows purification of 20 mg of prolidase in each preparation. The purity of the prolidase was found to be more than 99% when tested on polyacrylamide electrophoresis, and it showed homogeneity in crossed immunoelectrophoresis. The enzyme has a specific activity of about 200 units of activity per mg protein when determined against l -alanyl- l -proline. The specificity of the enzyme is in accordance with the original definition of an imidodipeptidase, as its activity is limited to dipeptides with the constitution aminoacyl- l -proline or aminoacyl- l -hydroxyproline, and it has no activity against tripeptides.

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.

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.

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.

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 β‐globin gene. These data demonstrate that the post‐weaning decline of LPH is mainly due to a transcriptional down‐regulation.