Phorbol 12-myristate 13-acetate-induced ectodomain shedding and phosphorylation of the human meprinbeta metalloprotease.

Shedding of proteins localized at the cell surface is an important regulatory step in the function of many of these proteins. Human meprin (N-benzoyl-l-tyrosyl-p-aminobenzoic acid hydrolase, PPH, EC 3.4.24.18) a zinc-metalloendopeptidase of the astacin family is an oligomeric protein complex of alpha- and beta-subunits and is expressed abundantly in the intestine and kidney as well as in leukocytes of the lamina propria and in cancer cells. In transfected cells intracellular proteolytic removal of the membrane anchor results in the secretion of the meprin alpha-subunit. In rats and mice, the beta-subunit exists in a membrane-anchored form. In contrast, human meprinbeta is constitutively converted into a secretable form. We now show that phorbol 12-myristate 13-acetate (PMA) stimulates an increased release of hmeprinbeta from transfected COS-1 cells, whereas hmeprinalpha secretion is not influenced. This stimulatory effect is inhibited by the protein kinase C (PKC) inhibitor staurosporine, suggesting that activation of PKC mediates PMA-induced hmeprinbeta shedding. The use of different protease inhibitors shows that two different metalloprotease activities are responsible for the constitutive and the PMA-stimulated hmeprinbeta shedding. We identified tumor necrosis factor alpha-converting enzyme (TACE or ADAM17) as the protease that mediates the PMA-induced release. We also demonstrate that hmeprinbeta is phosphorylated by PMA treatment on Ser687 within a PKC consensus sequence in the cytosolic domain of the protein. This phosphorylation of hmeprinbeta is not, however, implicated in the enhanced secretion by PMA treatment.

Disaccharidase activities in dyspeptic children: biochemical and molecular investigations of maltase-glucoamylase activity.

BACKGROUND: Maltase-glucoamylase enzyme plays an important role in starch digestion. Glucoamylase deficiency is reported to cause chronic diarrhea in infants, but its role in dyspeptic children is unknown. METHODS: Glucoamylase and other disaccharidase specific activities were assayed from duodenal biopsy specimens in 44 children aged 0.5-18 years (mean, 10 +/- 5 years) undergoing endoscopy to evaluate dyspeptic symptoms. All subjects had normal duodenal histology. Intestinal organ culture was used to evaluate synthesis and processing of maltase-glucoamylase. Sequencing of the maltase-glucoamylase coding region was performed in subjects with low activity or variation of isoform in organ culture. RESULTS: Twenty-two of the dyspeptic children had one or more disaccharidases with low specific activity. Twelve subjects (28%) had low activity of glucoamylase. Eight subjects had low activities of glucoamylase, sucrase, and lactase. Low glucoamylase activity was not correlated with the isoform phenotype of maltase-glucoamylase as described by metabolic labeling and sodium dodecyl sulfate electrophoresis. Novel nucleotide changes were not detected in one subject with low glucoamylase activity or in two subjects with variant isoforms of maltase-glucoamylase peptides. CONCLUSION: Twelve of 44 dyspeptic children had low specific activity of duodenal maltase-glucoamylase. Eight of these children had low specific activity of all measured disaccharidases.

Congenital maltase-glucoamylase deficiency associated with lactase and sucrase deficiencies.

BACKGROUND: Multiple enzyme deficiencies have been reported in some cases of congenital glucoamylase, sucrase, or lactase deficiency. Here we describe such a case and the investigations that we have made to determine the cause of this deficiency. METHODS AND RESULTS: A 2.5 month-old infant, admitted with congenital lactase deficiency, failed to gain weight on a glucose oligomer formula (Nutramigen). Jejunal mucosal biopsy at 4 and 12 months revealed normal histology with decreased maltase-glucoamylase, sucrase-isomaltase, and lactase-phlorizin hydrolase activities. Testing with a C-starch/breath CO loading test confirmed proximal starch malabsorption. Sequencing of maltase-glucoamylase cDNA revealed homozygosity for a nucleotide change (C1673T) in the infant, which causes an amino acid substitution (S542L) 12 amino acids after the N-terminal catalytic aspartic acid. The introduction of this mutation into "wildtype" N-terminus maltase-glucoamylase cDNA was not associated with obvious loss of maltase-glucoamylase enzyme activities when expressed in COS 1 cells and this amino-acid change was subsequently found in other people. Sequencing of the promoter region revealed no nucleotide changes. Maltase-glucoamylase, lactase, and sucrase-isomaltase were each normally synthesized and processed in organ culture. CONCLUSIONS: The lack of evidence for a causal nucleotide change in the maltase-glucoamylase gene in this patient, and the concomitant low levels of lactase and sucrase activity, suggest that the depletion of mucosal maltase-glucoamylase activity and starch digestion was caused by shared, pleiotropic regulatory factors.