Molecular cloning and expression of an alternative hKLK3 transcript coding for a variant protein of prostate-specific antigen.
We report the molecular cloning of a full-length cDNA corresponding to a 2.1-kb hKLK3 mRNA. This mRNA results from the alternative splicing of intron 4, and its accumulation in prostatic LNCaP cells is stimulated by androgen. The cDNA encodes a prepro-prostate-specific antigen (PSA) variant containing 238 amino acids. The new protein, PSA-related protein 1 (PSA-RP1), differs from PSA at the COOH-terminal end and lacks the serine residue that is essential for catalytic activity. Prepro-PSA-RP1 was transiently expressed in COS1 cells fused to the V5 epitope of the paramyxovirus SV5. The recombinant fusion protein was detected in the spent medium by Western blot analysis using anti-V5 and anti-PSA antibodies. This indicates that PSA-RP1 is secreted and has PSA-like antigenic epitopes. A pro-PSA and a pro-PSA-RP1 having a mutated propeptide were overproduced in Escherichia coli fused to glutathione S-transferase. The recombinant PSA and PSA-RP1 were matured in vitro and identified by Western blot with molecular masses of 29 (PSA) and 27 (PSA-RP1) kDa. The data indicate that PSA-RP1, not complexed to serine protease inhibitors, could be present in biological fluids, thus contributing to the free PSA-immunoreactive fraction in serum.
Heuzé
N
N
Laboratoire d'Enzymologie et Chimie des Protéines, UPRES-EA 2637, Université F. Rabelais, Tours, France.
Olayat
S
S
Gutman
N
N
Zani
M L
ML
Courty
Y
Y
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D003001:Cloning, Molecular; D003429:Cross Reactions; D006801:Humans; D008969:Molecular Sequence Data; D017430:Prostate-Specific Antigen / Q000235:genetics* / Q000276:immunology; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D017430:Prostate-Specific Antigen