Human and rat gamma glutamyl hydrolase antibodies

#The present invention relates generally to the peptidase gamma glutamyl hydrolase, and more particularly to nucleic acid molecules encoding gamma glutamyl hydrolase and uses thereof.

#Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.

#Gamma glutamyl hydrolase (GH) (EC 3.4.22.12) catalyzes the hydrolysis of the polyglutamate side-chain folyl polyglutamates and antifolyl polyglutamates (Stokstad and Koch 1967; Elsenhans et al. 1984; Chandler et al. 1986; Silink et al. 1975; Horne et al. 1981; Samuels et al. 1986; Bhandari et al. 1990; Rosenberg and Neuman 1974; Priest et al. 1982; Saini and Rosenberg 1974; Shin et al. 1974; McGuire and Coward 1984; Wang et al. 1986; Rao and Noronha 1977; Reisenauer et al. 1977; Wang et al. 1993). Gamma glutamyl hydrolase has been characterized from a number of sources, and it exhibits either endo- or exo-peptidase activity, depending upon the tissue of origin (Stokstad and Koch 1967; Elsenhans et al. 1984; Silink et al. 1975; Horne et al. 1981; Samuels et al. 1986; Bhandari et al. 1990; Rosenberg and Neuman 1974; Priest et al. 1982; Saini and Rosenberg 1974; Shin et al. 1974). In many tissues the enzyme is lysosomal with an acidic pH optimum (McGuire and Coward 1984). In addition, the enzyme is often sulfhydryl- and Zn.sup.2+ -dependent (Chandler et al. 1986; Silink et al. 1975; Horne et al. 1981; Bhandari et al. 1990; McGuire and Coward 1984; Wang et al. 1986; Rao and Noronha 1977; Wang et al. 1993), appears to be a glycoprotein in many cases (Silink et al. 1975; Wang et al. 1993), and has a reported molecular weight of 50 to 150 kDa (Elsenhans et al. 1984; Chandler et al. 1986; Silink et al. 1975; Priest et al. 1982; McGuire and Coward 1984; Rao and Noronha 1977; Reisenauer et al. 1977; Wang et al. 1993). A thorough analysis of the mechanism of this unique peptidase that cleaves only gamma glutamyl linkages is not available and a knowledge of the detailed structure of the protein and the gene encoding it are not yet delineated.

#Cloning and Sequencing of Rat GH

#The sequence of the open reading frame corresponding to gamma glutamyl hydrolase was determined using a combination of reverse transcription polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends (RACE), and screening of a commercial cDNA library. Total cellular RNA was prepared from rat hepatoma H35 cells using TRI REAGENT (Molecular Research Center, Inc., Cincinnati Ohio), according to the manufacturer's instructions. Poly(A).sup.+ mRNA was isolated from total RNA by affinity chromatography using a spin column of microcrystalline oligo(dT) cellulose (New England Biolabs, Beverly Mass.). Poly(A).sup.+ mRNA eluate was precipitated with ethanol and stored at -70.degree. C. for later use. The poly(A).sup.+ mRNA from H35 cells was reverse transcribed into cDNA using MMLV-reverse transcriptase and an oligo(dT) primer (Stratagene, Palo Alto, Calif.). The cDNA was subjected to PCR amplification using degenerate primers derived from the amino acid sequence of the N-terminus and of CNBr generated fragments. The primers used for RT-PCR were modified by carrying deoxyinosine residues at positions corresponding to ambiguous nucleotides (Ohtsuka et al. 1985). The RT-PCR generated a 605-base fragment denoted pGH-1.

#The cDNA prepared from H35 mRNA was synthesized by MMLV reverse-transcriptase and ligated to the Marathon cDNA adaptor (Clontech, Palo Alto, Calif.). RACE ready cDNA was amplified with nested GH-specific primers in combination with Marathon adaptor primers, AP1 and AP2, respectively. For sequencing, PCR products generated with Taq polymerase were cloned into a PCR II vector in the TA Cloning system, as the 3' A overhangs are not removed (Invitrogen, San Diego, Calif.). The amount of PCR product needed to ligate with 50 ng of PCR II vector was estimated according to the manufacturer's directions.