Roger H. Erickson

Identification and Characterization of Brush-Border Membrane-Bound Neutral Metalloendopeptidases From Rat Small Intestine

Neutral metalloendopeptidase enzymes were identified and partially characterized in the brush-border membranes of rat small intestinal mucosal cells using insulin B chain and glutaryl-trialanine-4-methoxy-beta-naphthylamide as substrates. Three different molecular species of endopeptidase were identified by disc gel electrophoresis. These enzymes were shown to be distinct from pancreatic endopeptidases on the basis of the following: enrichment in the brush-border membrane fraction, site of hydrolysis of peptide substrates, sensitivity to specific proteinase inhibitors, and the presence of brush-border membrane-associated endopeptidase activity in mucosal cells of Thirty-Vella loops. Hydrolysis of the substrates was shown to be a two-step process involving initial cleavage by endopeptidase with secondary hydrolysis of the peptide products by brush-border membrane aminopeptidase N. Hydrolysis of both substrates was maximum at a neutral pH and was strongly inhibited by metal chelating agents, phosphoramidone, and amastatin. Intestinal perfusion studies using glutaryl-trialanine-4-methoxy-beta-naphthylamide suggest that these enzymes play a physiologic role in protein digestion. It was concluded that neutral endopeptidases are integral components of the intestinal brush-border membrane and work in concert with aminopeptidase N to hydrolyze dietary protein. This process may be of nutritional importance in normal subjects and those with diminished exocrine pancreatic function.

Identification of upstream stimulatory factor as an activator of the human dipeptidyl peptidase IV gene in Caco-2 cells.

The 5 upstream region (-448/-443) of the human dipeptidyl peptidase IV gene promoter containing a consensus E-box (CACGTG) was shown to bind upstream stimulatory factor using nuclear extracts from mouse (3T3) fibroblasts and the human intestinal and hepatic epithelial cell lines Caco-2 and HepG2. Supershift analysis with specific antibodies to USF-1 and USF-2 indicates that USF-1 is the primary isoform binding to the E-box in nuclear extracts from these cells. Using cell culture, transient cotransfection of USF expression vectors with dipeptidyl peptidase IV promoter constructs revealed that both USF-1 and USF-2 caused an approximately tenfold increase in reporter gene expression in Caco-2 cells. Mutant forms of USF-1 and -2 lacking the DNA binding or transcriptional activation domains were unable to stimulate reporter gene expression. Mutation of the E-box prevented binding of USF, although stimulation of reporter gene expression by cotransfection with USF was reduced by only 50%. By using a series of deletion constructs in cotransfection experiments, a second possible site of USF interaction with the dipeptidyl peptidase IV promoter was localized to the -119/-88 region.

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.

Transcriptional regulation of rat intestinal angiotensin-converting enzyme and dipeptidyl peptidase IV by a high proline diet☆

Abstract A high proline diet (50% gelatin) was found to induce a 3–8 fold increase in the activity of rat small intestinal angiotensin-converting enzyme and dipeptidyl peptidase IV. Induction was also accompanied by a increase in steady state levels of enzyme mRNA. It was observed that the rate of gene transcription for these two enzymes is increased to levels corresponding to the changes in their mRNA. These results indicate for the first time that dietary components can influence the expression of various intestinal brush border membrane hydrolases at the level of gene transcription.

Localization and characterization of rat small intestinal aminopeptidase P and its role in prolyl peptide digestion

Abstract The distribution of aminopeptidase P was examined in rat small intestinal epithelial cells using the substrate Gly-Pro-Hyp. Soluble activity accounted for 65% of the total activity in these cells while 27% was membrane bound and localized to the brush border membrane fraction. Soluble and brush border membrane associated activities differed with respect to their pH optima, kinetic constants, and thermostability indicating that they are due to different enzymes. Both activities, however, exhibited similar sensitivity to several types of inhibitors. Brush border membrane aminopeptidase P was readily inhibited by metal chelating agents and was reactivated by addition of increasing concentrations of Mn2+. The mode of membrane association was investigated by detergent solubilization and enzymatic release of aminopeptidase P. A phosphatidylinositol-specific phospholipase C was the most effective in releasing aminopeptidase P, suggesting that the enzyme is anchored via a glycosyl-phosphatidylinositol moiety similar to alkaline phosphatase. These results indicate that aminopeptidase P is a major intestinal brush border membrane enzyme and probably plays an important role in conjunction with other intestinal prolyl peptidases in the digestion of proline containing peptides and proteins.

Analysis of dipeptidyl peptidase IV gene regulation in transgenic mice: DNA elements sufficient for promoter activity in the kidney, but not the intestine, reside on the proximal portion of the gene 5'-flanking region.

The dipeptidyl peptidase IV (DPPIV) gene encodes a brush border membrane exopeptidase that is expressed in a tissue‐restricted fashion. To examine the regulation of DPPIV transcription in various tissues in vivo, we examined the expression of DPPIV 5′‐flanking region (promoter)–human growth hormone reporter constructs in transgenic mice. These mice exhibited cell‐type specific reporter expression in kidney. Surprisingly, however, only very low to non‐detectable levels of reporter were found in small intestine. These results indicate that DNA elements sufficient for DPPIV expression in kidney, but not intestine, reside in the 5′‐flanking region of the gene.