Involvement of Glutamine-238 in the Substrate Specificity of Human Laeverin/Aminopeptidase Q

Abstract

Aminopeptidases hydrolyze the N-terminal amino acid of proteins or peptide substrates. Among them, the M1 family of zinc aminopeptidases (gluzincins) shares the consensus GAMEN and HEXXH(X) 18 E motifs essential for enzymatic activity. This family, which consists of 11 enzymes in humans, [1][2][3] plays important roles in several pathophysiological processes, such as angiogenesis, cell cycle regulation, reproduction, memory retention, blood pressure control, and antigen presentation to major histocompatibility complex (MHC) class I molecules. [4][5][6][7][8][9][10][11][12] Laeverin was originally identified as a cell-surface protein specifically expressed on human extravillous trophoblasts. 13) cDNA cloning of human laeverin revealed that it contains both consensus motifs and thus is a novel member of the M1 family. Another group also predicted the existence of the 'leaverin' gene in the human genome through a genomic search and named it aminopeptidase Q (APQ).14) Recently, we established a large-scale production system of the enzyme and characterized its enzymatic properties in detail. 15) We found that the enzyme degraded several placenta-derived peptide hormones, such as angiotensin III, endokinin C, and kisspeptin-10. Furthermore because its exopeptidase motif GAMEN is uniquely composed of the HAMEN sequence, we examined the enzymatic significance of His-379 of human laeverin/APQ by site-directed mutagenesis and found that this residue contributes to the unique properties of the enzyme.16) Considering the susceptibility of these peptides and their specific expression in the placenta, we speculated that laeverin/APQ plays important roles in the maintenance of normal pregnancy in humans.In our previous work, we identified a residue affecting enzymatic activity and the substrate specificity of endoplasmic reticulum aminopeptidase (ERAP)1, a member of the M1 aminopeptidase family. 17) In particular, replacement of Gln-181 with Asp caused a marked change in the substrate specificity of ERAP1. On the other hand, replacement with Ala led to almost complete loss of enzymatic activity. To further analyze the residue affecting enzymatic activity and substrate specificity of the M1 family of aminopeptidases, we examined in the present study the effect of replacing Gln-238 of human laeverin/APQ with Ala. We found that Q238A laeverin/APQ retained substantial enzymatic activities with marked change of its substrate specificity, indicating the significance of this residue for the enzymatic characteristics of laeverin/APQ. Data presented in this study imply that direct electrostatic interaction is not involved in the mutant enzyme's preference for basic amino acids, which was suggested in the previous work. 17) MATERIALS AND METHODS Molecular Modeling of Laeverin/APQThe published X-ray crystallographic structure of Thermoplasma acidophilum Tricorn-interacting factor F3 (TIFF3) (Protein Data Bank code 1Z5H) 18) was used as a template for modeling the catalytic site of ERAP1 with the three-dimensional Jigsaw Protein Server (h...

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“…Σν ERAP1 θαίλεηαη λα ζπκβάιιεη θαη ζηελ εγγελή αλνζναπφθξηζε ελεξγνπνηψληαο ηα καθξνθάγα. Σν ERAP1 δείρζεθε λα εθθξίλεηαη απφ ηα καθξνθάγα κεηά απφ ηελ επαγσγή ηνπο κε LPS/IFN-γ απμάλνληαο ηε θαγνθπηηαξηθή ηνπο δξαζηηθφηεηα [126]. Σν έλδπκν πνπ εθθξηλφηαλ είρε κνξηαθφ βάξνο 114kDa δηαθέξνληαο απφ απηφ πνπ θαηαθξαηείηαη ζην ΔΓ (105kDa) θαη δηάζεηε ζχλζεηνπο Ν-νιηγνζαθραξίηεο πνπ επηβεβαηψλνπλ ηε ζσζηή έθθξηζε ηνπ ελδχκνπ κέζσ ηνπ Golgi.…”

“…Δθηφο απφ ην ERAP1, άιιεο δχν ακηλνπεπηηδάζεο, ην IRAP θαη ην laeverin/APQ, δείρζεθαλ λα απμάλνπλ ηε θαγνθπηηαξηθή δξαζηηθφηεηα ησλ καθξνθάγσλ θαη αθνχ θαη απηέο είλαη ακηλνπεπηηδάζεο ιεπθίλεο είλαη πνιχ πηζαλφ ε δξαζηηθφηεηα ακηλνπεπηηδάζεο ιεπθίλεο λα είλαη ζεκαληηθή γηα ηελ επαγσγή ηεο θαγνθχησζεο. Απηή ε ππφζεζε εληζρχεηαη απφ ην γεγνλφο φηη ηα καθξνθάγα παξνπζίαδαλ απμεκέλε θαγνθχησζε είηε επσάδνληαλ κε ζξεπηηθφ πιηθφ πνπ πεξηείρε ERAP1 είηε κε ζξεπηηθφ πιηθφ ην νπνίν είρε θηιηξαξηζηεί γηα λα απνκαθξπλζεί ην ERAP1 [126]. Δίλαη επνκέλσο πηζαλφ ην ERAP1 λα είλαη ππεχζπλν γηα ηελ παξαγσγή ζπγθεθξηκέλσλ πεπηηδίσλ ηα νπνία επάγνπλ ηε θαγνθχησζε.…”