Human meprin beta: O-linked glycans in the intervening region of the type I membrane protein protect the C-terminal region from proteolytic cleavage and diminish its secretion.

Human meprin (hmeprin; N -benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase; EC 3.4.24.18) is a member of the astacin family of zinc metalloendopeptidases. The major site of expression is the brush border membrane of small intestinal and kidney epithelial cells. The enzyme is a type I integral membrane protein composed of two distinct subunits, alpha and beta, which are linked by disulphide bridges. The enzyme complex is attached to the plasma membrane only via the beta-subunit. The alpha-subunit is cleaved in the endoplasmic reticulum in a constitutive manner to remove the C-terminal membrane anchor which leads to secretion of the protein. While the beta-subunit of hmeprin remains largely attached to the brush-border membrane some proteolytic processing occurs intracellularly as well as at the cell surface and results in the release of this subunit from the cell. In the present paper, we report that the beta-subunit bears multiple O-linked sugar residues in the intervening domain. In contrast, the alpha-subunit does not contain O-linked oligosaccharides. Our results show that the O-linked carbohydrate side chains in hmeprinbeta are clustered around a 13 amino acid sequence that contains the main cleavage site for proteolytic processing of the subunit. Prevention of O-glycosylation by specific inhibitors leads to enhanced proteolytic processing and the consequence is an increased release of hmeprinbeta into the culture medium.

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.