Point mutations abolishing the mannose-binding capability of boar spermadhesin AQN-1 Mahnaz Ekhlasi-Hundrieser a,b, ⁎, Juan J. Calvete c, ⁎, Bettina Von Rad a , Christiane Hettel a,b , Manfred Nimtz d , Edda Töpfer-Petersen a a Department of Reproductive Biology, University of Veterinary Medicine Hannover, Foundation, Bünteweg 2, 30559 Hannover, Germany b Current address: Clinic for Cattle, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, 30173 Hannover, Germany c Instituto de Biomedicina de Valencia, C.S.I.C, Valencia, Spain d Helmholtz Centre for Infection Research (HZI), Inhoffenstraße 7, 38124 Braunschweig, Germany article info abstract Article history: Received 5 January 2008 Received in revised form 11 February 2008 Accepted 19 February 2008 Available online 4 March 2008 The mannose-binding capability of recombinant wild-type boar spermadhesin AQN-1 and of its site-directed mutants in the highly-conserved region around of the single glycosylation site (asparagine 50) of some spermadhesins, where the carbohydrate binding site has been proposed to be located, was checked using a solid-phase assay and a biotinylated mannose ligand. Substitution of glycine 54 by amino acids bearing an unipolar side chain did not cause significant decrease in the mannose-binding activity. However, amino acids with uncharged polar side chains or having a charged polar side chain abolished the binding of biotinylated mannose to the corresponding AQN-1 mutants. The results suggest that the higher surface accessibility of amino acids possessing polar side chains compared to those bearing nonpolar groups may sterically interfere with monosaccharide binding. The location of the mannose-binding site in AQN-1 appears to be topologically conserved in other heparin-binding boar spermadhesins, i.e., AQN-3 and AWN, but departs from the location of the mannose-6-phosphate-recognition site of PSP-II. This indicates that different spermadhesin molecules have evolved non-equivalent carbohydrate-binding capabilities, which may underlie their distinct patterns of biological activities. © 2008 Elsevier B.V. All rights reserved. Keywords: Seminal plasma protein Spermadhesin AQN-1 Acidic seminal fluid Protein Carbohydrate-binding protein CUB domain 1. Introduction Mammalian sperm do not possess the ability to fertilize the egg immediately upon ejaculation, but acquire fertilization competence following residence in the female's reproductive tract for a certain period of time. The length of time required varies with species, but usually takes several hours. This time-dependent sperm maturational process, known as “capacitation”, was first observed in 1951 inde- pendently by M.C. Chang [1] and C.R. Austin [2], and endow sper- matozoa to undergo stimulated acrosome reaction. The seminal plasma (SP), constituted by secretions from the testes, epididymis and the accessory sex glands, contains a variety of factors (amino acids, lipids, fatty acids, osmolytes, peptides, and proteins) that influence the viability of ejaculated spermatozoa and the ability of acrosome-intact sperm to undergo the acrosome reaction in response to the oocyte's zona pellucida [3–5]. Thus, the seminal plasmas of a variety of mammalian species contain both factors that prevent inappropriate acrosome reactions (decapacitating factors) and pro- teins that upon binding to the sperm surface enhance the fertilizing potential of spermatozoa [6–8]. The emerging view is that the mem- brane remodeling events that underlie capacitation of ejaculated spermatozoa in the female's genital tract is essentially conserved though it can be accomplished by different seminal plasma proteins in different species [9]. Hence, the effects of seminal plasma proteins on homologous sperm capacitation may represent an example of functional convergence of distinct molecular players. The major seminal plasma proteins of ungulates (i.e., pig, bull, ram, stallion) belong to the spermadhesin family and to the Fn2- choline-binding proteins [10–12]. Spermadhesins are male secretory proteins detected so far in ungulates (pig, cattle, horse and ram) [13– 16]. In the pig, this family of proteins comprises five members, AQN-1, AQN-3, AWN, PSP-I and PSP-II, whose genes are clustered on SCC 14q28-q29 [15]. Together they represent over 90% of the total boar seminal plasma proteins [17] and are thought to participate in different aspects of porcine fertilization. Porcine spermadhe- sins, 110–133-residue polypeptides, are synthesized by the epididy- mis and accessory glands [18]. Spermadhesins are multifunctional proteins exhibiting different sperm-coating and ligand-binding cap- abilities, and their specific pattern of biological activities is modu- lated by sequence variation, glycosylation, and the aggregation state [10,19–24]. Biochimica et Biophysica Acta 1784 (2008) 856–862 ⁎ Corresponding authors. M. Ekhlasi-Hundrieser is to be contacted at Clinic for Cattle, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, 30173 Hannover, Germany. Fax: +49 511 953 827373. J. J. Calvete, Instituto de Biomedicina de Valencia, C.S.I.C, Valencia, Spain. Fax: +34 96 369 0800. E-mail addresses: mahnaz.ekhlasi@tiho-hannover.de (M. Ekhlasi-Hundrieser), jcalvete@ibv.csic.es (J.J. Calvete). 1570-9639/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2008.02.015 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap