740
V.-L. Pham et al. / Biochimie 93 (2011) 730e741
[6] G. Fontes, A.D. Lajoix, F. Bergeron, S. Cadel, A. Prat, T. Foulon, R. Gross, S. Dalle,
D. Le-Nguyen, F. Tribillac, D. Bataille, Miniglucagon-generating endopeptidase,
which processes glucagon into miniglucagon, is composed of NRD convertase
and aminopeptidase B, Endocrinology 146 (2005) 702e712.
[7] D. Bataille, Pro-protein convertases in intermediary metabolism: islet
hormones, brain/gut hormones and integrated physiology, J. Mol. Med. 85
(2007) 673e684.
[8] V. Hook, S. Yasothornsrikul, D. Greenbaum, K.F. Medzihradszky, K. Troutner,
T. Toneff, R. Bundey, A. Logrinova, T. Reinheckel, C. Peters, M. Bogyo, Cathepsin
L and Arg/Lys aminopeptidase: a distinct prohormone processing pathway for
the biosynthesis of peptide neurotransmitters and hormones, Biol. Chem. 385
(2004) 473e480.
[9] M.C. Beinfeld, L. Funkelstein, T. Foulon, S. Cadel, K. Kitagawa, T. Toneff,
T. Reinheckel, C. Peters, V. Hook, Cathepsin L plays a major role in cholecys-
tokinin production in mouse brain cortex and in pituitary AtT-20 cells:
protease gene knockout and inhibitor studies, Peptides 10 (2009) 1882e1891.
[10] V. Hook, L. Funkelstein, T. Toneff, C. Mosier, S.R. Hwang, Human pituitary
contains dual cathepsin L and prohormone convertase processing pathway
components involved in converting POMC into the peptide hormones ACTH,
alpha-MSH, and beta-endorphin, Endocrine 35 (2009) 429e437.
[11] T. Foulon, S. Cadel, V. Chesneau, M. Draoui, A. Prat, P. Cohen, Two novel
metallopeptidases with a specificity for basic residues. Functional properties,
structure and cellular distribution, Ann. N.Y. Acad. Sci. 780 (1996) 106e120.
[12] S. Cadel, T. Foulon, A. Viron, A. Balogh, S. Midol-Monnet, N. Noel, P. Cohen,
Aminopeptidase B from the rat testis is a bifunctional enzyme structurally
related to leukotriene-A4 hydrolase, Proc. Natl. Acad. Sci. U.S.A. 94 (1997)
2963e2968.
[13] A. Balogh, S. Cadel, T. Foulon, R. Picart, A. Der Garabedian, A. Rousselet,
C. Tougard, P. Cohen, Aminopeptidase B: a processing enzyme secreted and
associated with the plasma membrane of rat pheochromocytoma (PC12) cells,
J. Cell Sci. 111 (1998) 161e169.
[14] G. Thoidis, T. Kupriyanova, J.M. Cunningham, P. Chen, S. Cadel, T. Foulon,
P. Cohen, R. Fine, K.V. Kandror, Glucose transporter Glut3 is targeted to
secretory vesicles in neurons and PC12 cells, J. Biol. Chem. 274 (1999)
14062e14066.
substrate [51]. Moreover, the binding pocket is hydrophobic and
narrow in order to fit the aliphatic chain. This might also explain
why LTA4H and Ap-B share common characteristics. LTA4H
catalyzes LTA4 in LTB4 and is also able to process basic amino acids
presenting an aliphatic side chain. In contrast with Ap-B, the
aminopeptidase specificity of the LTA4H is larger, able to cleave
substrates such as Arg, Lys, Ala, Leu and Pro [52,53] suggesting local
reorientation and/or specific amino acid interactions in the S1
pocket allowing these cleavages. Results concerning the substrate
specificity of G298P indicate that local rearrangements induced in
the b-sheet of the GXMEN motif could reorganize the active site and
lead to a favourable position of hydrophobic residues such as Ala,
Leu or Pro. The side chain of Proline residue is bonded both to the
amino group and to the a-carbon leading to a cyclic structure. This
cyclic structure induces important constraints on the conformation
of the polypeptide backbone. The bond most likely affected in
G298P is the Phe297eCOeNHePro298 bond. The mutation could
induce a different orientation of the side chain of the Phe residue
and a steric hindrance in the S1 pocket implicated in the binding
of the P1 residue of the substrate. As there is no difference in the
GXMEN motif of Ap-B and LTA4H, their differences in substrate
specificity do not only depend on this motif. Contrary to the
other members of the M1 family, Ap-B and LTA4H exhibit a peculiar
GGMEN motif with two Gly residues implicated in hydrogen bonds
with the peptidic bond of the substrate. Interestingly, a Tyr residue
is present in LTA4H just upstream this GGMEN signature, while it is
a Phe residue in Ap-B. Therefore, it will be interesting to mutate this
amino acid into a Tyr residue to verify if there is some difference
in the specificity of Ap-B activity. Indeed, recent studies have
shown that the corresponding Ala residue in IRAP primary structure
is implicated in substrate and inhibitor specificity [54]. All these
observations will provide some insights into the different substrate
specificity of the M1 family aminopeptidases. Further experiments
are necessary to encompass the complex role of this GXMEN
motif in the catalytic mechanism of the proteolysis of physiological
substrates by Ap-B and other M1 family members.
[15] C. Piesse, M. Tymms, E. Garrafa, C. Gouzy, M. Lacasa, S. Cadel, P. Cohen, T. Foulon,
Human aminopeptidase B (rnpep) on chromosome 1q32.2: complementary
DNA, genomic structure and expression, Gene 292 (2002) 129e140.
[16] C. Piesse, S. Cadel, C. Gouzy-Darmon, J.C. Jeanny, V. Carrière, D. Goidin, L. Jonet,
D. Gourdji, P. Cohen, T. Foulon, Expression of aminopeptidase
B in the
developing and adult rat retina, Exp. Eye Res. 79 (2004) 639e648.
[17] J.Z. Haeggström, F. Tholander, A. Wetterholm, Structure and catalytic
mechanisms of leukotriene A4 hydrolase, Prostaglandins Other Lipid Mediat.
83 (2007) 198e202.
[18] N.D. Rawlings, A.J. Barrett, Evolutionary families of peptidases, Biochem. J. 290
(1993) 205e218.
[19] N.M. Hooper, Families of zinc metalloproteases, FEBS Lett. 354 (1994) 1e6.
[20] N.D. Rawlings, F.R. Morton, C.Y. Kok, J. Kong, A.J. Barrett, MEROPS: the
peptidase database, Nucleic Acids Res. 36 (2007) D320e325.
Acknowledgments
[21] T. Foulon, S. Cadel, P. Cohen, Molecules in Focus: Aminopeptidase
B
(EC 3.4.11.6), Int. J. Biochem. Cell Biol. 31 (1999) 747e750.
[22] L. V-Pham, S. M-Cadel, C. Gouzy-Darmon, C. Hanquez, M.C. Beinfeld, P. Nicolas,
C. Etchebest, T. Foulon, Aminopeptidase B, a glucagon-processing enzyme: site
directed mutagenesis of the Zn2þ-binding motif and molecular modeling,
BMC Biochem. 8 (2007) 21.
[23] M.M. Thunnissen, P. Nordhund, J.Z. Häeggstrom, Crystal structure of human
leukotriene A4 hydrolase, a bifunctional enzyme in inflammation, Nat. Struct.
Biol. 8 (2001) 131e135.
This work was supported by funds from the University Pierre et
Marie Curie (UPMC, ER3), the University Denis Diderot (UMR S
665), the Institut National de la Santé et de la Recherche Médicale
(INSERM, UMR S 665) and the Institut National de Transfusion
sanguine (INTS, UMR S 665).
We thank Drs S. Fermandjian, L. Zargarian (UMR 8113 CNRS,
Villejuif France) and C. El Amri (UR4 UPMC, Paris, France) for
their help in circular dichroism and helpful discussions, and Drs
D. Deville-Bonne and D. Topalis (ER3 UPMC, Paris, France) for their
help in fluorescence spectroscopy.
[24] B.W. Matthews, Structural basis of the action of thermolysin and related zinc
peptidases, Acc. Chem. Res. 21 (1988) 333e340.
[25] F. Tholander, A. Muroya, B.P. Roques, M.C. Fournié-Zaluski, M.M. Thunnissen,
J.Z. Haeggström, Structure-based dissection of the active site chemistry of
leukotriene A4 hydrolase: implications for M1 aminopeptidases and inhibitor
design, Chem. Biol. 15 (2008) 920e929.
[26] R. S-Hwang, A. O’Neill, S. Bark, T. Foulon, V. Hook, Secretory vesicle amino-
peptidase B related to neuropeptide processing: molecular identification and
subcellular localization to enkephalin- and NPY-containing chromaffin
granules, J. Neurochem. 100 (2007) 1340e1350.
References
[27] N. Luciani, C. Marie-Claire, E. Ruffet, A. Beaumont, B.P. Roques, M.C. Fournié-
[1] V. Chesneau, A.R. Pierotti, N. Barré, C. Créminon, C. Tougard, P. Cohen, Isolation
and characterization of a dibasic selective metalloendopeptidase from rat
testis that cleaves at the aminoterminus of arginine residues, J. Biol. Chem.
269 (1994) 2056e2061.
[2] A.V. Azaryan, V.Y.H. Hook, Unique cleavage specificity of “prohormone
thiol protease” related to proenkephalin processing, FEBS Lett. 341 (1994)
197e202.
[3] S. Cadel, A.R. Pierotti, T. Foulon, C. Creminon, N. Barré, D. Segretain, P. Cohen,
Aminopeptidase-B in the rat testis: isolation, functional properties and
cellular localization in the seminiferous tubules, Mol. Cell. Endocrinol. 110
(1995) 149e160.
Zaluski, Characterization of Glu350 as
a critical residue involved in the
N-terminal amine binding site of aminopeptidase N (EC 3.4.11.2): insights
into its mechanism of action, Biochemistry 37 (1998) 686e692.
[28] G. Vazeux, X. Iturrioz, P. Corvol, C. Llorens-Cortes,
A glutamate residue
contributes to the exopeptidase specificity in aminopeptidase A, Biochem. J.
334 (1998) 407e413.
[29] X. Iturrioz, R. Rozenfeld, A. Michaud, P. Corvol, C. Llorens-Cortes, Study of
asparagine 353 in aminopeptidase A: characterization of
a novel motif
(GXMEN) implicated in exopeptidase specificity of monozinc aminopepti-
dases, Biochemistry 40 (2001) 14440e14448.
[30] P.G. Laustsen, S. Vang, T. Kristensen, Mutational analysis of the active site of
human insulin-regulated aminopeptidase, Eur. J. Biochem. 268 (2001) 98e104.
[31] P.C. Rudberg, F. Tholander, M.M. Thunnissen, J.Z. Haeggstrom, Leukotriene
[4] T. Foulon, S. Cadel, A. Prat, V. Chesneau, V. Hospital, D. Segretain, C. Tougard,
P. Cohen, NRD convertase and Aminopeptidase B: two putative processing
metallopeptidases with a selectivity for basic residues, Ann. Endocrinol. 58
(1997) 357e364.
[5] S. Cadel, C. Piesse, C. Gouzy-Darmon, P. Cohen, T. Foulon, Aminopeptidase
B: from protein to gene, Curr. Top. Pept. Prot. Res. 6 (2004) 37e45.
A4 hydrolase/aminopeptidase. Glutamate 271 is
a catalytic residue with
specific roles in two distinct enzyme mechanisms, J. Biol. Chem. 277 (2002)
1398e1404.