Journal of Chromatography B, 876 (2008) 198–202 Contents lists available at ScienceDirect Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb Purification and identification of transglutaminase from mouse coagulating gland and its cross-linking activity among seminal vesicle secretion proteins Huan-Chin Tseng a,1 , Han-Jia Lin b,1 , P.S. Sudhakar Gandhi a , Chia-Yih Wang a , Yee-Hsiung Chen a,c,∗ a Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan b Institute of Bioscience and Biotechnology, Center for Marine Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan c Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan article info Article history: Received 15 August 2008 Accepted 26 October 2008 Available online 31 October 2008 Keywords: Coagulating gland Protein cross-link Protein identification Seminal coagulation Seminal vesicle Transglutaminase abstract A 75-kDa protein secreted from mouse coagulating gland was purified to homogeneity by a series of isolation steps including ion exchange chromatography on a DEAE-Sephacel column and ion exchange high-performance liquid chromatography on a sulfopropyl column. It was identified to be Type IV transg- lutaminase (TG 4 ), based on the establishment of N-terminal sequences by automated Edman degradation together with partial sequences by MS analysis. Its cross-linking activity was tested on the reduced sample of mouse seminal secretion which contained seven major monomer proteins tentatively designated as SVS I–VII. The enzyme was able to cross-link any of SVS I–III but failed to cross-link the other SVS proteins with a M r value less than 14kDa. SVS I and SVS III showed comparable substrate activity, but were much weaker than SVS II during the TG 4 catalysis. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Upon ejaculation, seminal vesicle secretion (SVS) constitutes the major portion of seminal plasma that is coagulated in a substan- tial number of mammalian species, including many myomorphic rodents, some moles, hedgehogs, marsupials, rabbits, stallions, boars and several primates [1]. The deposition of semen coagu- lum in animals such as rodents into the vagina at coitus results in the formation of a copulatory plug that occludes the vaginal barrel close to the uterine cervix. It should be noted that extirpation of seminal vesicle and coagulating gland from mice and rats prevents formation of the copulatory plug and this results in greatly reduced fertility [2,3], manifesting the indispensible roles of these two male accessory sexual glands in seminal coagulation. Transglutaminase (TG; EC2.3.2.13) catalyzes protein cross-links via isopeptide formation [4]. This enzymatic reaction is generally Abbreviations: A25 peptide, biotin-TVQQEL; BPNH2, 5-(biotinamido) pentylamine; CGS, coagulating gland secretion; DTT, dithiothreitol; GST, glu- tathione S-transferase; PBST, phosphate buffer saline containing 0.1% Tween 20; PMSF, phenylmethylsulphonyl fluoride; SVS, seminal vesicle secretion; TG4, type IV transglutaminase. ∗ Corresponding author at: Institute of Biological Chemistry, Academia Sinica, P.O. Box 23-106, Taipei 106, Taiwan. Fax: +886 2 2363 5038. E-mail address: bc304@gate.sinica.edu.tw (Y.-H. Chen). 1 Equal contributions. believed to be essential for the formation of a semisolid gelati- nous mass in human semen [5,6] or the seminal clotting in rodent semen [7,8]. Such enzyme activity has been illustrated in the human prostate [9] and in rat coagulating gland secretions (CGS) [10]. Although it has been shown that incubation of mouse SVS with TG of guinea pig liver (TG 2 ) result in protein cross-linking [8,11], TG 2 is not the actual enzyme involved in seminal coagulation during natural coitus. Rather, TG 4 from male sexual gland is responsible for this reproductive event. Therefore, it becomes a pre-requisite to purify TG 4 in order to study how it cross-links the SVS proteins. Mice are good experimental animals for this study from two view points. Firstly, purification of TG 4 from CGS in quantity is feasible. Secondly, some progress has been made on the analysis of the mouse SVS proteins that had been shown to consist of several minor proteins such as SVA [12], P12 [13], Ceacam 10 [14], and seven well-resolved monomer proteins designated SVS I–VII in the decreasing order of M r values (95,000–8000) according to their mobility on reduced SDS–PAGE [15,16]. These results are an added advantage to identify the TG 4 substrates in the SVS. This work aims to purify and identify TG 4 from mouse CGS, and determine its protein substrates among mouse SVS proteins. 2. Materials and methods The following materials were obtained from commercial sources: DEAE-Sephacel (Amersham Corp., Buckinghamshire, UK 1570-0232/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2008.10.041