Epididymal lipocalin-type prostaglandin D2 synthase: identification using mass spectrometry, messenger RNA localization, and immunodetection in mouse, rat, hamster, and monkey.
This study identified prostaglandin D2 synthase (PGDS) in murine epididymal fluid using a proteomic approach combining two-dimensional (2D) gel electrophoresis and mass spectrometry (MS). The caudal epididymal fluid was collected by retroperfusion, and proteins were separated by 2D gel electrophoresis followed by matrix-assisted laser desorption ionization MS analyses after trypsin digestion. The identification was based on the protein-specific peptide map as well as on sequence information generated by nano-electrospray ionization MS/MS. By in situ hybridization, the mRNA was detected in caput, corpus, and cauda, but it was not detected in the initial segment. The PGDS protein was mostly detected in the corpus and cauda by Western blot analysis and immunohistochemistry using a specific polyclonal antibody. In caudal fluid, PGDS was distributed among several isoforms (pI range, 6.5-8.8), suggesting that this protein undergoes posttranslational modification of its primary sequence. After N-glycanase digestion, the molecular mass decreased from 20-25 to 18.5 kDa, its theoretical mass. The PGDS was also detected in the epididymis of rat, hamster, and cynomolgus monkey from the caput to the cauda. In conclusion, MS is a powerful and accurate technique that allows unambiguous identification of the murine epididymal PGDS. The protein is 1) present throughout the epididymis, except in the initial segment, with an increasing luminal concentration from distal caput to cauda; 2) a major protein in caudal fluid; 3) an N-glycosylated, highly polymorphic protein; and 4) conserved during evolution.
Fouchécourt
Sophie
S
Department of Obstetrics and Gynecology, Biochemistry, Urologic Surgery, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
Chaurand
Pierre
P
DaGue
Beverly B
BB
Lareyre
Jean-Jacques
JJ
Matusik
Robert J
RJ
Caprioli
Richard M
RM
Orgebin-Crist
Marie-Claire
MC
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D006224:Cricetinae; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004822:Epididymis / Q000033:anatomy & histology / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D007202:Indicators and Reagents; D019746:Intramolecular Oxidoreductases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D054834:Lipocalins; D008252:Macaca fascicularis; D008297:Male; D008647:Mesocricetus; D051379:Mice; D008969:Molecular Sequence Data; D020543:Proteome / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D013045:Species Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D007202:Indicators and Reagents; D007527:Isoenzymes; D054834:Lipocalins; D020543:Proteome; D012333:RNA, Messenger; D006026:Glycoside Hydrolases; C020668:glycanase; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase