ACE2, the first known human homologue of angiotensin-converting enzyme (ACE), was identified from 5' sequencing of a human heart failure ventricle cDNA library. ACE2 has an apparent signal peptide, a single metalloprotease active site, and a transmembrane domain. The metalloprotease catalytic domains of ACE2 and ACE are 42% identical, and comparison of the genomic structures indicates that the two genes arose through duplication. In contrast to the more ubiquitous ACE, ACE2 transcripts are found only in heart, kidney, and testis of 23 human tissues examined. Immunohistochemistry shows ACE2 protein predominantly in the endothelium of coronary and intrarenal vessels and in renal tubular epithelium. Active ACE2 enzyme is secreted from transfected cells by cleavage N-terminal to the transmembrane domain. Recombinant ACE2 hydrolyzes the carboxy terminal leucine from angiotensin I to generate angiotensin 1-9, which is converted to smaller angiotensin peptides by ACE in vitro and by cardiomyocytes in culture. ACE2 can also cleave des-Arg bradykinin and neurotensin but not bradykinin or 15 other vasoactive and hormonal peptides tested. ACE2 is not inhibited by lisinopril or captopril. The organ- and cell-specific expression of ACE2 and its unique cleavage of key vasoactive peptides suggest an essential role for ACE2 in the local renin-angiotensin system of the heart and kidney. The full text of this article is available at http://www. circresaha.org.

Human angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a zinc metalloprotease whose closest homolog is angiotensin I-converting enzyme. To begin to elucidate the physiological role of ACE2, ACE2 was purified, and its catalytic activity was characterized. ACE2 proteolytic activity has a pH optimum of 6.5 and is enhanced by monovalent anions, which is consistent with the activity of ACE. ACE2 activity is increased ϳ10-fold by Cl ؊ and F ؊ but is unaffected by Br ؊ . ACE2 was screened for hydrolytic activity against a panel of 126 biological peptides, using liquid chromatographymass spectrometry detection. Eleven of the peptides were hydrolyzed by ACE2, and in each case, the proteolytic activity resulted in removal of the C-terminal residue only.

The TAPR locus containing the TIM gene family is implicated in the development of atopic inflammation in mouse, and TIM-1 allelic variation has been associated with the incidence of atopy in human patient populations. In this study, we show that manipulation of the TIM-1 pathway influences airway inflammation and pathology. Anti-TIM-1 mAbs recognizing distinct epitopes differentially modulated OVA-induced lung inflammation in the mouse. The epitopes recognized by these Abs were mapped, revealing that mAbs to both the IgV and stalk domains of TIM-1 have therapeutic activity. Unexpectedly, mAbs recognizing unique epitopes spanning exon 4 of the mucin/stalk domains exacerbated immune responses. Using Ag recall response studies, we demonstrate that the TIM-1 pathway acts primarily by modulating the production of TH2 cytokines. Furthermore, ex vivo cellular experiments indicate that TIM-1 activity controls CD4+ T cell activity. These studies validate the genetic hypothesis that the TIM-1 locus is linked to the development of atopic disease and suggest novel therapeutic strategies for targeting asthma and other atopic disorders.

Lipoprotein lipase (LPL) is central to triglyceride metabolism. Severely compromised LPL activity causes familial chylomicronemia syndrome (FCS), which is associated with very high plasma triglyceride levels and increased risk of life-threatening pancreatitis. Currently, no approved pharmacological intervention can acutely lower plasma triglycerides in FCS. Low yield, high aggregation, and poor stability of recombinant LPL have thus far prevented development of enzyme replacement therapy. Recently, we showed that LPL monomers form 1:1 complexes with the LPL transporter glycosylphosphatidylinositol-anchored high-density lipoprotein–binding protein 1 (GPIHBP1) and solved the structure of the complex. In the present work, we further characterized the monomeric LPL/GPIHBP1 complex and its derivative, the LPL–GPIHBP1 fusion protein, with the goal of contributing to the development of an LPL enzyme replacement therapy. Fusion of LPL to GPIHBP1 increased yields of recombinant LPL, prevented LPL aggregation, stabilized LPL against spontaneous inactivation, and made it resistant to inactivation by the LPL antagonists angiopoietin-like protein 3 (ANGPTL3) or ANGPTL4. The high stability of the fusion protein enabled us to identify LPL amino acids that interact with ANGPTL4. Additionally, the LPL–GPIHBP1 fusion protein exhibited high enzyme activity in in vitro assays. Importantly, both intravenous and subcutaneous administrations of the fusion protein lowered triglycerides in several mouse strains without causing adverse effects. These results indicate that the LPL–GPIHBP1 fusion protein has potential for use as a therapeutic for managing FCS.

Through an integrated study of the reactivity of a monoclonal antibody, 803-15.6, with synthetic peptides and native recombinant HIV-1 envelope glycoprotein gp120, we have obtained structure-functional information on a region of rgp120 not yet elucidated by X-ray crystallography. mAb 803-15.6 binds with high affinity and broad cross-clade specificity to the conserved C-terminal region (amino acids 502-516) of HIV-1 rgp120. Phage display selection from a random peptide library identified the core binding motif as AXXKXRH, homologous to residues 502-508. Using quantitative binding analyses, the affinity of mAb 803-15.6 for native, monomeric recombinant gp120HXB2 (rgp120) was found to be similar to that for the synthetic gp120 peptide (502-516). Circular dichroism studies indicate that the synthetic peptide largely has a random coil conformation in solution. The results therefore suggest that the 803-15.6 epitope is fully accessible on rgp120 and that this region of rgp120 is as flexible as the synthetic peptide. Residues 502-504 are on the edge of a putative gp41 binding site that has been postulated to change conformation on CD4 binding. However, the affinity of mAb 803-15.6 for rgp120 is not affected by binding of CD4 and vice-versa. These results suggest either that the 502-504 region does not change conformation upon CD4 binding, or that recombinant gp120 does not undergo the same changes as occur in the native viral gp120-gp41 oligomer. The detailed characterization of the 803-15.6 epitope may be useful for further study of the role of the C5 region of gp120 in the viral attachment and fusion process.