COOH-terminal proteolytic processing of secreted and membrane forms of the alpha subunit of the metalloprotease meprin A. Requirement of the I domain for processing in the endoplasmic reticulum.
Cell surface isoforms of meprin A (EC 3.4.24.18) from mice and rats contain beta subunits that are type I integral membrane proteins and alpha subunits that are disulfide-linked to or noncovalently associated with membrane-anchored meprin subunits. Both alpha and beta subunits are synthesized with COOH-terminal domains predicted to be cytoplasmic, transmembrane, and epidermal growth factor-like; these domains are retained in beta subunits but are removed from alpha during maturation. The present studies establish that an inserted 56-amino acid domain (the "I" domain), present in alpha but not in beta, is necessary and sufficient for COOH-terminal proteolytic processing of the alpha subunit. This was demonstrated by expression of mutant meprin subunits (deletion mutants, chimeric alpha beta subunits, and beta mutants containing the I domain) in COS-1 cells. Mutations of two common processing sites present in the I domain (a dibasic site and a furin site) did not prevent COOH-terminal proteolytic processing, indicating that the proteases responsible for cleavage are distinct from those having these specificities. Deletion of the I domain from the alpha subunit resulted in accumulation of unprocessed subunits in a preGolgi compartment. Furthermore, COOH-terminal proteolytic processing of wild-type alpha subunits occurred before acquisition of endoglycosidase H resistance. Pulse-chase experiments and expression of an alpha subunit transcript containing a c-myc epitope tag, confirmed that proteolytic processing at the COOH terminus occurs in the endoplasmic reticulum. This work identifies the region of the alpha subunit that is essential for COOH-terminal processing and demonstrates that the differential processing of the evolutionarily-related subunits of meprin A that results in a structurally unique tetrameric protease begins in the endoplasmic reticulum.
Marchand
P
P
Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey 17033.
Tang
J
J
Johnson
G D
GD
Bond
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002522:Chlorocebus aethiops; D010450:Endopeptidases / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006026:Glycoside Hydrolases; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007668:Kidney; D046911:Macromolecular Substances; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013462:Sulfur Radioisotopes; D014162:Transfection
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D008715:Methionine; D006026:Glycoside Hydrolases; D010450:Endopeptidases; D008666:Metalloendopeptidases; C053833:meprin A