Human aminopeptidase P gene

Details Human aminopeptidase P gene Human aminopeptidase P gene

1998-09-02

2002-06-04

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S069100, C435S252300, C435S320100, C435S471000, C435S006120, C536S023200

Reexamination Certificate

active

06399349

ABSTRACT:

BACKGROUND OF THE INVENTION
Evidence of an aminoacylproline hydrolase was first encountered in studies of the metabolism of bradykinin (BK). It was found that BK (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) is inactivated virtually quantitatively during a single passage through the rat pulmonary vascular bed (1,2). BK is degraded through 5-8 half-lifes during the 2-3 sec required for a single transit from the right to the left side of the heart (31). If, as appears to be the case, the overall metabolism occurs within the pulmonary capillary bed (mean transit time of about 0.2 sec), the half-life of BK within the capillary bed is on the order of 0.03 sec. From these data, it was postulated that the relevant kininase enzymes are situated on, or near, the luminal surface of pulmonary endothelium so as to have access to intravascular substrates (1,2,4). In time, it was shown that angiotensin converting enzyme (ACE) plays a central role in the inactivation of BK and is, in fact, disposed on the luminal surface of pulmonary microvascular endothelium (5,6). ACE was found to account for one of the hydrolytic reactions (cleavage of the Pro
7
-Phe
8
bond) observed in the earliest studies (2).
The original data indicated that a peculiar aminopeptidase also participated in the degradation of BK (1,2). The result caused some concern and confusion in that none of the aminopeptidases then known was capable of hydrolyzing an imido bond (Arg
1
-Pro
2
). Shortly thereafter, an aminoacylproline hydrolase was isolated from an extract of
E. coli
and was shown to be capable of hydrolyzing polyproline and the Arg
1
-Pro
2
bond of BK (204,205). The bacterial enzyme was named aminopeptidase P, a name now used for aminoacylproline hydrolases obtained from any animal or plant source.
Shortly after the discovery of
E. coli
AmP, it was found that pig kidney extracts contained a particulate-associated AmP activity and that the AmP-like substance was not solubilized by detergents (78). The AmP-like material, believed to have been solubilized in a butanol/aqueous solvent system, behaved like a complex mixture of substances on chromatography. As a further complication, pig kidney AmP did not hydrolyze polyproline, the substrate used to assay
E. coli
AmP. A weakly reactive synthetic substrate was prepared, Gly-Pro-Hyp, and AmP activity was measured in terms of the rate of formation of free glycine in a two-step assay protocol (78).
Membrane-associated AmP remained effectively inaccessible to conventional chemical and biochemical analysis until the early 1990's. The difficulties of AmP catalytic assay was solved by preparing the synthetic substrate Arg-Pro-Pro-[
3
H]benzylamide (APPBz-
3
H) (20 Ci/mmol), a substrate related to the N-terminal tripeptide of bradykinin (21,22). APPBz-
3
H proved to be highly reactive with AmP and could, by virtue of its high specific radioactivity, be used under conditions of first order enzyme kinetics. However, the problem of the inefficient solubilization of particulate-associated AmP was not solved, and a search for soluble forms of AmP was therefore conducted. Guinea pig serum was found to be an enormously rich source of AmP (21,22) and was used as the starting material to obtain apparently homogeneous AmP in two isoforms, Mr 89,000 and 81,500 (22).
Independently, Hooper et al (111) solved the problem of solubilizing pig kidney AmP. They found that AmP is bound to membranes via a glycosyl phosphatidylinositol (GPI) lipid anchor and can be solubilized efficiently using phosphatidylinositol-specific phospholipase C (PI-PLC). Subsequently, Simmons et al (180) and Ryan, et al. (32) showed that rat and bovine lung and guinea pig lung and kidney forms of AmP are solubilized by PI-PLC. Human kidney AmP is also solubilized by PI-PLC. Once thus solubilized, AmP no longer behaves anomalously on conventional chromatography matrices.
Aminopeptidase P (AmP; EC 3.4.11.9) is the only known human enzyme capable of hydrolyzing a N-terminal imido bond, a bond common to many collagen degradation products and some neuropeptides, cytokines and vasoactive peptides (14,16,21,22,31,98,111,146,147,152,165,192,205). AmP occurs in cell membrane-bound and intracellular soluble forms and is not uniformly distributed among tissues nor among cell-types of a given tissue (21,39,165,205), which implies that physiologic roles of AmP are determined by anatomic disposition (a determinant of reaction conditions and access to substrates) as well as by catalytic selectivity.
It is therefore an object of the present invention to help define both molecular and anatomic determinants of AmP functions.
SUMMARY OF THE INVENTION
The genomic DNA and full-length cDNA sequence of human kidney AmP has been determined. The deduced amino acid sequence indicates that AmP is a member of the recently-recognized “pita bread-fold” protein family, a family of very little sequence homology but of high similarity in three-dimensional structure (59). Within the “pita bread-fold” family, there is a subdivision called the “proline peptidase” family, with which human kidney AmP shares at least five short blocks of amino acid sequences of fair to high homology (although overall homologies are low). These blocks are known to contain the amino acid residues that compose the catalytic site of
E. coli
methionine aminopeptidase, a metallo-peptidase whose structure has been determined by x-ray crystallography (59). Based on these comparisons, it is postulated that human kidney AmP amino acid residue H430 serves as the proton shuttle, and D450, D461, H520, E555 and E569 (see SEQ ID NO:2) are the catalytic metal ligands. This can be tested by preparing the site-specific mutants H430F, D450N, D461N, H520F, E555Q and E569Q. In addition, each of five potential N-glycosylation sites and each of five C residues can be mutated to examine for indirect effects of glycosyl groups and disulfide bonds on catalytic activity, solubility and protein stability. In addition, one can determine the chromosome location of AmP.
Using the sequence and immunocytochemistry at the level of electron microscopy (EM), one can define, in major organs, the cellular and subcellular sites of AmP, and, using subcellular fractions, dispositions of AmP in terms of anatomically proximate receptors and cell signaling molecules (the bradykinin B2 receptor, eNOS and guanylate cyclase) whose activities may directly or indirectly be affected by AmP activities.
This will help characterize structure-function relationships of human AmP at three levels; 1. molecular structure/catalytic activity, 2. cellular and subcellular distributions that determine orientations (and access to substrates) of the catalytic site, and 3. disposition in respect to “nearest-neighbor” effector and cell signaling molecules.
In addition to providing conceptual advances in understanding of AmP functions, this work provides tools (antibodies and oligonucleotide probes) useful for clinical studies of AmP deficiency states.
Use of the cDNA, genomic DNA, or a combination, for protein expression has commercial implications. The inferred amino acid sequence can be used as a starting point for defining higher structure and function. Through protein expression, crystals can be prepared for determination of higher structure. Reverse transcriptase-polymerase chain reactions was used to obtain four overlapping fragments of AmP cDNA. The intact full-length cDNA can be obtained by ligation. The first (nt 1-474) and second (359-734) fragments are digested with XmnI (nt 365) and then ligated. The product (1-734) and the third fragment (634-1702) are digested with SacI (nt 652) and ligated to yield 1-1702; which, with the fourth fragment (1588-3428), are digested with ScaI (nt 1625) and ligated to yield 1-3428. DNA encoding human AmP can also be produced by direct synthesis of appropriate oligonucleotides based on the disclosed amino acid and nucleotide sequences. For large scale protein expression, the full-length DNA is transferred into the expression vector pVL1393 and used with co-transfectant, Baculogold,

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