The angiotensin-converting enzyme (ACE)-related carboxypeptidase, ACE2, is a type I integral membrane protein of 805 amino acids that contains one HEXXH ؉ E zincbinding consensus sequence. ACE2 has been implicated in the regulation of heart function and also as a functional receptor for the coronavirus that causes the severe acute respiratory syndrome (SARS). To gain further insights into this enzyme, the first crystal structures of the native and inhibitor-bound forms of the ACE2 extracellular domains were solved to 2.2-and 3.0-Å resolution, respectively. Comparison of these structures revealed a large inhibitor-dependent hinge-bending movement of one catalytic subdomain relative to the other (ϳ16°) that brings important residues into position for catalysis. The potent inhibitor MLN-4760 ((S,S)-2-{1-carboxy-2-[3-(3,5-dichlorobenzyl)-3H-imidazol4-yl]-ethylamino}-4-methylpentanoic acid) makes key binding interactions within the active site and offers insights regarding the action of residues involved in catalysis and substrate specificity. A few active site residue substitutions in ACE2 relative to ACE appear to eliminate the S 2 substrate-binding subsite and account for the observed reactivity change from the peptidyl dipeptidase activity of ACE to the carboxypeptidase activity of ACE2.The angiotensin-converting enzyme (ACE) 1 -related carboxypeptidase, ACE2, is a type I integral membrane protein of 805 amino acids that contains one HEXXH ϩ E zinc-binding consensus sequence (1, 2). The catalytic domain of ACE2 is 42% identical to that of its closest homolog, somatic angiotensinconverting enzyme (sACE; EC 3.4.15.1), a peptidyl dipeptidase that plays an important role in the renin angiotensin system for blood pressure homeostasis. The loss of ACE2 in knockout mice has no effect on blood pressure, but reveals ACE2 as an essential regulator of heart function (3). In a recent discovery, ACE2 was identified as a functional receptor for the coronavirus that is linked to the severe acute respiratory syndrome (SARS) (4, 5).The physiological differences observed in the phenotypes of ACE (6, 7) and/or ACE2 (3) knockout mice presumably reflect the significant differences in substrate specificity and reactivity between these enzymes. Many substrates for ACE2 were identified by screening biologically active peptides (8). In all cases, only carboxypeptidase activity was found. Of the seven best in vitro peptide substrates identified (k cat /K m Ͼ 10 5 M Ϫ1 s Ϫ1 ), proline and leucine are the preferred P 1 residues, with a partiality for hydrophobic residues in the P 1 Ј position, although basic residues at P 1 Ј are also cleaved (peptide-binding subsites in proteins are as previously defined (9)). Some of the best in vitro peptide substrates are apelin-13, des-Arg 9 -bradykinin, angiotensin II, and dynorphin A-(1-13). The longest peptide substrate identified is a 36-residue peptide, apelin-36 (8). An examination of the ACE2 and ACE literature may be found in recently published reviews (10 -12).We report here the first crystal ...
Flt4 is a receptor protein tyrosine kinase that is expressed in the adult lymphatic endothelium and high endothelial venules. We have used a BIAcore assay to identify rodent and human cell conditioned media containing the ligand of Flt4 (Flt4-L). Receptor-based anity chromatography was used to purify this growth factor, followed by amino acid sequencing and molecular cloning of the murine cDNA, the orthologue of human vascular endothelial growth factor-C and vascular endothelial growth factor related protein. The murinē t4-L gene was localized to chromosome 8 and demonstrated to be widely expressed. Flt4-L was found to have a hydrophobic signal sequence and a pro-peptidelike sequence that is removed to generate the mature Nterminus. In addition, the C-terminal region of Flt4-L has four repeats of a cysteine-rich motif that is presumably also proteolytically processed to generate the 21 000 M r polypeptide subunit of the Flt4-L homodimer. Recombinant Flt4-L activated Flt4 as judged by induction of tyrosyl phosphorylation, and induced mitogenesis in vitro of lymphatic endothelial cells.Keywords: Flt4; Flt4 ligand; VEGF-C; BIAcoreTo identify a source of the Flt4 ligand, we used a Flt4-Fc fusion protein. This consists of the extracellular region of murine Flt4 (Finnerty et al., 1993) fused to the hinge-C H 2-C H 3 domains of human immunoglobulin g1. The conditioned media (CM) of over 100 dierent cell lines were screened for binding to Flt4-Fc using a BIAcore 2000 instrument. The media conditioned by the murine embryonic liver cell line, BNL 1NG A.2, human bladder carcinoma 5637, and bualo rat BRL 3A each showed binding to Flt4-Fc, but not human IgG1 (Figure 1). The binding was speci®c because when the CM was coinjected with excess Flt4-Fc, but not human IgG1 or KIT-Fc, the signal was inhibited (Figure 1). The presence of Flt4 ligand (Flt4-L) in BNL 1NG A.2 CM was con®rmed by demonstrating that it activated the Flt4 receptor protein tyrosine kinase. CHO cells stably transfected with a murine Flt4 cDNA expression vector were treated with 50-fold concentrated BNL 1NG A.2 CM for 10 min at 378C. This resulted in a marked increase in tyrosyl phosphorylation of Flt4 (Figure 2a, lane 1) relative to mock stimulated samples (Figure 2a, lane 2).Six micrograms of Flt4-L were puri®ed from 15L of BNL 1NG A.2 CM using Flt4-Fc anity chromatography. Flt4-Fc (100 mg/ml) or KIT-Fc (200 mg/ml) were directly immobilized to 1 mL Pharmacia HiTrap NHS columns. A volume of 50 ml of concentrated BNL 1NG A.2 CM was loaded ®rst onto the KIT-Fc column to adsorb nonspeci®c binding, then onto the Flt4-Fc column. The columns were washed and separately eluted with 24 mM hydrochloric acid. As expected, only fractions from the Flt4-Fc anity column had binding to Flt4-Fc on the BIAcore assay. The speci®c activity in response units (RU)/mg was determined using RU that were in the linear range of RU versus concentration of Flt4-L puri®cation fractions. The speci®c activity of Flt4-L from BNL 1NG A.2 was 4.2610 6 RU/mg, an increase of 98 455-fold after t...
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