Cloning and characterization of canine prostate-specific membrane antigen.
BACKGROUND
Prostate-specific membrane antigen (PSMA) is a promising biomarker in the diagnosis of prostate cancer and a potential target for antibody-based therapeutic strategies. We isolated the canine PSMA cDNA and investigated the cellular and biochemical characteristics of the recombinant protein as a potential target for animal preclinical studies of antibody based-therapies.


METHODS
Canine PSMA cDNA was isolated by PCR, cloned into expression vectors and transfected into COS-1 and MDCK cells. The biosynthesis and glycosylation of the recombinant protein were investigated in pulse-chase experiments, the cellular localization by confocal laser microscopy, the mode of association of PSMA with the membrane with solubilization in different detergents and its quaternary structure in sucrose-density gradients.


RESULTS
Canine PSMA shows 91% amino acid homology to human PSMA, whereby the major difference is a longer cytoplasmic tail of canine PSMA compared to its human counterpart. Canine PSMA is trafficked efficiently along the secretory pathway, undergoes homodimerization when it acquires complex glycosylated mature form. It associates with detergent-resistant membranes, which act as platforms along its intracellular trafficking. Confocal analysis revealed canine PSMA at the cell surface, Golgi, and the endoplasmic reticulum. A similar distribution is revealed for human PSMA, yet with reduced cell surface levels.


CONCLUSIONS
The cloning, expression, biosynthesis, processing and localization of canine PSMA in mammalian cells is described. We demonstrate that canine PSMA reveals similar characteristics to human PSMA rendering this protein useful as a translational model for investigations of prostate cancer as well as a suitable antigen for targeted therapy studies in dogs.
Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Hannover, Germany.
Sonja
S
Schmidt
Giulio
G
Fracasso
Marco
M
Colombatti
Hassan Y
HY
Naim
D000595:Amino Acid Sequence; D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods*; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D043425:Glutamate Carboxypeptidase II / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D008297:Male; D008969:Molecular Sequence Data; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000378:metabolism; D020836:Protein Structure, Quaternary; D021381:Protein Transport / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014162:Transfection; D057170:Translational Research, Biomedical
D000954:Antigens, Surface; D014408:Biomarkers, Tumor; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II