Identification of a novel human peroxisomal 2,4-dienoyl-CoA reductase related protein using the M13 phage protein VI phage display technology.
Recently, we reported the successful use of the gVI-cDNA phage display technology to clone cDNAs coding for novel peroxisomal enzymes by affinity selection using immobilized antisera directed against peroxisomal subfractions (Fransen, M.; Van Veldhoven, P.P.; Subramani, S. Biochem. J., 1999, 340, 561-568). To identify other unknown peroxisomal enzymes, we further exploited this promising approach. Here we report the isolation and cloning of another novel human cDNA encoding a protein ending in the tripeptide AKL, a C-terminal peroxisomal targeting signal (PTS1). Primary structure analysis revealed that this molecule shared the highest sequence similarity to members of the 2,4-dienoyl-CoA reductase (DCR) family. However, functional analysis indicated that a recombinantly expressed version of the novel protein did not possess DCR activity with either 2-trans,4-trans-hexadienoyl-CoA or 2-trans,4-trans-decadienoyl-CoA as a substrate. The recombinant protein interacted with HsPex5p, the human PTS1-binding protein. Binding was competitively inhibited by a PTS1-containing peptide and was abolished when the last amino acid of the PTS1 signal was deleted. Transfection of mammalian cells with gene fusions between green fluorescent protein (GFP) and the human cDNA confirmed a peroxisomal localization and, therefore, the functionality of the PTS1. These results further demonstrate the suitability of the gVI-cDNA phage display technology for cDNA expression cloning using an antibody as a probe.
Amery
L
L
Departement Moleculaire Celbiologie, Katholieke Universiteit Leuven, Belgium.
Mannaerts
G P
GP
Subramani
S
S
Van Veldhoven
P P
PP
Fransen
M
M
D000595:Amino Acid Sequence; D000818:Animals; D017104:Bacteriophage M13 / Q000201:enzymology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D044943:Fatty Acid Desaturases / Q000378:metabolism*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D044925:Oxidoreductases Acting on CH-CH Group Donors; D020675:Peroxisomes / Q000201:enzymology*; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D014760:Viral Fusion Proteins / Q000378:metabolism*
C440769:M13 phage protein VI; D014760:Viral Fusion Proteins; D044943:Fatty Acid Desaturases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C100153:2,4-dienoyl-CoA reductase