Human meprinβ: 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

Abstract

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, α and β, which are linked by disulphide bridges. The enzyme complex is attached to the plasma membrane only via the β-subunit. The α-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 β-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 β-subunit bears raultiple O-linked sugar residues in the intervening domain. In contrast, the α-subunit does not contain O-linked oligosaccharides. Our results show that the O-linked carbohydrate side chains in hmeprinβ 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 hmeprinβ into the culture medium.