Protein disulfide isomerase-P5, down-regulated in the final stage of boar epididymal sperm maturation, catalyzes disulfide formation to inhibit protein function in oxidative refolding of reduced denatured lysozyme.
In mammalian spermiogenesis, sperm mature during epididymal transit to get fertility. The pig sharing many physiological similarities with humans is considered a promising animal model in medicine. We examined the expression profiles of proteins from boar epididymal caput, corpus, and cauda sperm by two-dimensional gel electrophoresis and peptide mass fingerprinting. Our results indicated that protein disulfide isomerase-P5 (PDI-P5) human homolog was down-regulated from the epididymal corpus to cauda sperm, in contrast to the constant expression of protein disulfide isomerase A3 (PDIA3) human homolog. To examine the functions of PDIA3 and PDI-P5, we cloned and sequenced cDNAs of pig PDIA3 and PDI-P5 protein precursors. Each recombinant pig mature PDIA3 and PDI-P5 expressed in Escherichia coli showed thiol-dependent disulfide reductase activities in insulin turbidity assay. Although PDIA3 showed chaperone activity to promote oxidative refolding of reduced denatured lysozyme, PDI-P5 exhibited anti-chaperone activity to inhibit oxidative refolding of lysozyme at an equimolar ratio. SDS-PAGE and Western blotting analysis suggested that disulfide cross-linked and non-productively folded lysozyme was responsible for the anti-chaperone activity of PDI-P5. These results provide a molecular basis and insights into the physiological roles of PDIA3 and PDI-P5 in sperm maturation and fertilization.
Akama
Kuniko
K
Graduate School of Science, Chiba University, Inage-Ku, Chiba 263-8522, Japan. akama@faculty.chiba-u.jp
Horikoshi
Tomoe
T
Sugiyama
Atsushi
A
Nakahata
Satoko
S
Akitsu
Aoi
A
Niwa
Nobuyoshi
N
Intoh
Atsushi
A
Kakui
Yasutaka
Y
Sugaya
Michiko
M
Takei
Kazuo
K
Imaizumi
Noriaki
N
Sato
Takaya
T
Matsumoto
Rena
R
Iwahashi
Hitoshi
H
Kashiwabara
Shin-ichi
S
Baba
Tadashi
T
Nakamura
Megumi
M
Toda
Tosifusa
T
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D015536:Down-Regulation / Q000502:physiology*; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004822:Epididymis / Q000201:enzymology; D005306:Fertilization / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D006801:Humans; D008297:Male; D018832:Molecular Chaperones / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D019704:Protein Disulfide-Isomerases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013045:Species Specificity; D013091:Spermatogenesis / Q000502:physiology*; D013094:Spermatozoa / Q000201:enzymology*; D013552:Swine
D004220:Disulfides; D004792:Enzyme Precursors; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D009113:Muramidase; D019704:Protein Disulfide-Isomerases