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 containi...

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Bibliographic Details
Published in:Cancer research (Chicago, Ill.) Ill.), 1999-06, Vol.59 (12), p.2820-2824
Main Authors: HEUZE, N, OLAYAT, S, GUTMAN, N, ZANI, M.-L, COURTY, Y
Format: Article
Language:English
Subjects:
Alternative Splicing
Amino Acid Sequence
Animals
Base Sequence
Biological and medical sciences
Cloning, Molecular
COS Cells
Cross Reactions
Host-tumor relations. Immunology. Biological markers
Humans
Medical sciences
Molecular Sequence Data
Prostate-Specific Antigen - genetics
Prostate-Specific Antigen - immunology
Recombinant Fusion Proteins - genetics
RNA, Messenger - analysis
RNA, Messenger - genetics
Tumor Cells, Cultured
Tumors
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Summary: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.
ISSN:0008-5472
1538-7445