title: Proteolytic fragmentation reveals the oligomeric and domain structure of porcine aminopeptidase A.
abstract: Aminopeptidase A (glutamyl aminopeptidase; EC 3.4.11.7) has been cloned from porcine brain and kidney cortex cDNA libraries and the complete primary sequence of the enzyme deduced. This predicts a type II integral membrane protein of 942 amino acids with 14 potential N-linked glycosylation sites and a His-Glu-Xaa-Xaa-His zinc binding motif. Aminopeptidase A was purified from porcine kidney cortex by a combination of anion exchange and hydrophobic interaction chromatographies following its release from the membrane by trypsin. The purified protein migrated as three major polypeptides on SDS-polyacrylamide gel electrophoresis of M(r) 147,000, 107,000, and 45,000. N-Terminal sequencing revealed that both the Mr 147,000 and 107,000 polypeptides had the same N-terminal sequence resulting from cleavage of aminopeptidase A by trypsin at the Lys-42-Asp-43 bond just outside the membrane-spanning hydrophobic region. Immunoelectrophoretic blot analysis following electrophoresis under nonreducing conditions revealed that the trypsin-cleaved form of the enzyme no longer migrated as a disulfide-linked dimer, placing the interchain disulfide link N-terminal to Lys-42. N-Terminal sequencing of the M(r) 45,000 polypeptide in the purified preparation of aminopeptidase A revealed that it resulted from cleavage at the Asn-602-Gly-603 bond by an endogenous protease. This posttranslational proteolytic cleavage occurred in porcine kidney cortex microvillar membranes but not in porcine intestinal microvillar membranes. Incubation of purified porcine kidney aminopeptidase N (membrane alanyl aminopeptidase; EC 3.4.11.2) with trypsin resulted in a similar fragmentation pattern to that observed in aminopeptidase A, suggesting that these and other members of the type II membrane-spanning zinc aminopeptidase family may have two distinct domains: an N-terminal domain, containing the zinc binding site and residues identified as being involved in catalysis, and a C-terminal domain of unknown function, that are separated by a protease-susceptible region.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Leeds, U.K.
authors.forename: J R
authors.initials: JR
authors.lastname: Hesp
authors.forename: N M
authors.initials: NM
authors.lastname: Hooper
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D018826:CD13 Antigens / Q000378:metabolism; D003001:Cloning, Molecular; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D043384:Glutamyl Aminopeptidase; D006031:Glycosylation; D007672:Kidney Cortex / Q000201:enzymology*; D008667:Metalloproteins / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational / Q000502:physiology; D017421:Sequence Analysis; D013552:Swine; D014357:Trypsin / Q000378:metabolism; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
chemical_list: D004220:Disulfides; D008667:Metalloproteins; D010446:Peptide Fragments; D000626:Aminopeptidases; D018826:CD13 Antigens; D043384:Glutamyl Aminopeptidase; D010196:Pancreatic Elastase; D014357:Trypsin; D015032:Zinc