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Isolation and Partial Characterisation of a Protein from Buck Seminal Plasma (Capra Hircus), Homologous to Spermadhesins

Authors:
Dárcio Ítalo Alves Teixeira at Universidade Estadual do Ceará
Dárcio Ítalo Alves Teixeira
Benildo Sousa Cavada at Universidade Federal do Ceará
Benildo Sousa Cavada
Alexandre Sampaio at Universidade Federal do Ceará
Alexandre Sampaio
Alexandre Havt at Universidade Federal do Ceará
Alexandre Havt
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Spermadhesins are a family of secretory proteins expressed in the male genital tract of pig, horse and bull. Their function and structure have been widely studied, especially those isolated from boar. However, there are no data concerning spermadhesins isolated from buck. Buck seminal plasma was collected and subjected to ion exchange chromatography on DEAE-Sephacel column followed by chromatography in a C18 column coupled to a HPLC system. The purification of the protein was determined by SDS-PAGE and MALDI-TOF analysis exhibiting a molecular mass of 12.5 KDa and showed to be structurally homologous to spermadhesins from boar and stallion.
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331
Protein and Peptide Letters, Vol. 9, No. 4, pp. 331-335, 2002
Bentham Science Publishers Ltd.
0929-8665/02 $ 35.00 + .00
ISOLATION AND PARTIAL CHARACTERISATION OF A
PROTEIN FROM BUCK SEMINAL PLASMA (Capra hircus),
HOMOLOGOUS TO SPERMADHESINS
D.I.A. Teixeira
1
, B.S. Cavada
2*
, A.H. Sampaio
3
, A. Havt
4
, C. Bloch Jr.
5
, M.V.Prates
5
, F.B.M.B. Moreno
2
,
E.A.Santos
1
, C.A.A.Gadelha
2
, T.S.Gadelha
2
, F.S.M.Crisóstomo
1
, V. J. F.
Freitas
1*
1
State University of Ceará, Laboratory of Physiology and Control of Reproduction, Fortaleza-CE-Brazil, 60740-000.
vjff@uece.br.
2
BioMol Lab – Department of Biochemistry and Molecular Biology – Federal University of
Ceará. Fortaleza, Ceará, Brazil. P.O. Box 6033. 60451-970. bscavada@ufc.br.
3
Department of Fishing
Engineering.
4
Department of Physiology and Pharmacology. Federal University of Ceará.
5
EMBRAPA/CENARGEN, Brasília, DF.
*Authors for correspondence
Abstract: Spermadhesins are a family of secretory proteins expressed in the male genital tract of pig, horse and bull. Their
function and structure have been widely studied, especially those isolated from boar. However, there are no data concerning
spermadhesins isolated from buck. Buck seminal plasma was collected and subjected to ion exchange chromatography on
DEAE-Sephacel column followed by chromatography in a C18 column coupled to a HPLC system. The purification of the
protein was determined by SDS-PAGE and MALDI-TOF analysis exhibiting a molecular mass of 12.5 KDa and showed to be
structurally homologous to spermadhesins from boar and stallion.
INTRODUCTION
Semen in mammals is composed of seminal plasma, secreted by accessory glands of the male reproductive
tract, and spermatozoa. According to [1] mammal’s seminal plasma exert many important effects in the
spermatozoa physiology and its protein composition varies from species to species. Recent report also indicates that
seminal plasma contains factors that influence both the fertilizing ability of spermatozoa and promotes important
effects on the female reproductive system [2].
A protein family named spermadhesins has been detected in boar, stallion [3] and bull [4]. This group of
proteins with low molecular weight (12-16 kDa) emerges as a novel protein family of animal lectins, which coat
332
the sperm surface at ejaculation and are believe to play major roles in sperm capacitation and gamete interactions
[2].
The amino acid sequences of spermadhesins determined until now have not shown any similarities with
known carbohydrate recognition domains (CRD). They belong to the superfamily of proteins with CUB domain,
with a predicted all-β structure.
The function and structure of spermadhesins have been widely studied, especially in boar. Porcine seminal
plasma contains five polypeptides, which were named AQN-1, AQN-3, AWN-I and AWN II, PSP-I and PSP-II, and
are the major secretory products of the seminal vesicle epithelium [5]. AWN-I is also synthesized by the rete testis
and tubuli recti [6] and is the only member of this family present in epididymal sperm. These peptides possess 110
to 133 amino acids residues, which are connected by two disulfide bridges and showed 40 to 60% of homology in
their primary structure [7].
After ejaculation 12-60 x 10
6
molecules of each of AQN-1, AQN-3, AWN (I and II), PSP-I and PSP-II
become coated on one third of the apical region of the acrosomal cap, but most of this coating material is released
during in vitro capacitation [8]. It was also suggested that AWN-I and AQN-3 molecules could form the first layer
of the coating material by interacting with the lipid bilayer. Fractionation of solubilized boar sperm membrane
proteins on immobilized porcine zona pellucida glycoproteins identified AWN-I and AQN-3 as the major zona
pellucida-binding proteins [9] indicating that these spermadhesins bind tightly to the sperm surface [10].
It has been described that a variety of mammalian species (pig, cat, rat, goat, horse, bull, rabbit and
human) contains decapacitation factors, which prevent inappropriate acrosome reactions, and that spermadhesins
have been responsible for this particulate function [3]. However, few data demonstrated the presence of
spermadhesins in the seminal plasma of buck. The aim of the present work is to isolate and characterize a possible
spermadhesin present in buck and to demonstrate its similarity with other proteins of the same family.
MATERIAL AND METHODS
Animals
Four 10 months old Saanen bucks were used for sample collection. Those animals were maintained in an
intensive system. They were fed with Pannisetum purpurem in the mornings and with concentrated forage (18% of
protein) in the afternoons. They had access to clean water and mineral licks ad libitum.
Semen collection
Semen collection was done twice a week always at seven in the morning by the use of an artificial vagina
associated with a graduated tube (mL). To help collection a buck female was used which had its estrus induced by
intramuscular inoculation of 1mL of estradiol cipionate (ECP
®
) weekly.
333
Semen conservation
The semen collected was placed in one Falcon tube and frozen. As soon as 10 mL of semen was collected
of each animal the tubes were defrosted and semen was transferred to Eppendorf tubes and centrifuged at 160 x g
for 10 minutes. The residues were discarded, and the supernatant collected, dialyzed against distilled water and
freeze-dried.
Detection and isolation
Freeze-dried materials were dissolved in 0.02M phosphate buffer, pH 6.2. The samples were applied to an
ion-exchange chromatography column (DEAE-Sephacel). After elution of the non-retained material the column was
eluted with a NaCl buffer gradient (0-1M). The main peak was applied to a C18 column coupled to an HPLC
system. The protein material collected in the HPLC was analysed by sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE) according to [11]. The material was then analysed by mass spectrometry (MS) using a
PE Biosystems Voyager DE-STR at a 25 kV in a linear mode, using α-cyano-4-dymetoxicinamic acid as matrix.
The N-terminal of this protein was sequenced on an Applied Biosystems 477A.
RESULTS AND DISCUSSION
Buck seminal plasma after ion exchange chromatography on a DEAE-Sephacel column (Figure 1),
followed by reverse phase HPLC (not shown), yielded a pure protein as demonstrated by PAGE-SDS (Figure 2 -
insert) with an apparent molecular mass of 1 2 . 0 0 0 Da. This new spermadhesin was named BSFP (buck seminal
fluid protein). This molecular mass was confirmed by mass spectrometry (MALDI-TOF), which exhibited a value
of 12.591 Da (Figure 2). These data were similar to that reported for AWN, a multifunctional protein with 14 kDa,
isolated from boar semen, which is the best structurally characterized spermadhesin so far [9]. Another member of
this protein family, named aSFP (acid seminal fluid protein), which is also a non-glucosylated protein with a low
molecular weight of 12.9 kDa, was purified from bull seminal plasma [12]. The N-terminal sequence of BSFP
determined by automatic protein sequence analysis is presented in Table 1. BSFP exhibited N-terminal sequence
homology to boar spermadhesins (AQN and AWN), and stallion spermadhesin (AWN) (Table 1). Reinert et al. [13]
demonstrated that AWN, isolated from boar and stallion, exhibited a high N-terminal sequence homology. AWN
from boar and stallion were shown to have the ability to bind zona pellucida glycoproteins. Since BSFP has
structure homology to those spermadhesins, we suggest that this protein may express the same biological activity,
binding to glycoproteins present in zona pellucida. Results on molecular mass similarity by SDS-PAGE and
MALDI-TOF, and N-terminal sequence homology strongly suggested that BSFP, this new protein, might belong to
the spermadhesin family. Furthermore, as has been reported for all spermadhesins isolated until now, no
haemagglutinating activity was expressed by BSFP. Further analysis must be carried out to better characterize the
structure and function of this new protein.
334
Figure 1 - Ion-exchange chromatography on DEAE-Sephacel of the seminal plasma from buck . The column was
washed with 0.02M phosphate buffer, pH 6.2, at flow rate of 30 mL.h
-1
and then eluted with linear gradient of 0 to
1M NaCl.
Figure 2 – MALDI-TOF/MS (Matrix-Assisted Laser Desorption Ionization) of buck spermadhesin. Insert: SDS-
polyacrylamide gel of electrophoresis: 1) and 5) boar spermadhesin (14 kDa); 2) buck spermadhesin (12,5 kDa); 3)
semen after DEAE chromatography; 4) buck seminal plasma.
1
1
2
3
4
1
0
0.5
1
1.5
2
2.5
1 11 21 31 41 51
Tubes
Absorbance (280nm)
0
1,0
0,2
0.4
0,6
0,8
2.5
2.0
0.5
1.5
1.0
0.8
0.2
0.6
NaCl (M )
335
Table 1 - N-terminal sequences from boar spermadhesins (AQN-I, AWN), stallion spermadhesin (AWN) and buck
spermadhesin.
Lectin N-terminal sequences
Buck
spermadhesin
ECDETRKCGGVHRDFSGRISCCF
10 20
AQN 1 boar AQNKGPHKCGGVLRNYSGRISTYEGPKTDCIWTILAKPGSRVFVAIPYLNLACGKEYVEV
10 20 30 40 50 60
AWN boar
AWNRRSRSCGGVLRDPPGKIFNSDGPQKDCVWTIKVKPHFHVVLAIPPLNLSCGKEYVEL
10 20 30 40 50 60
AWN stallion
AWNRRSRSCGGVLRDPPGKIFNSDGPQKDCVWTIKVKPHFHVVIAIPPLNLSCGKEYVEL
10 20 30 40 50 60
ACKNOWLEDGEMENTS:
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq),
Fundação Cearense de Amparo a Pesquisa (FUNCAP), Fundação Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES) and Programa de Apoio ao Desenvolvimento Científico e Tecnológico (PADCT). The
authors are grateful to Dr. Juan J. Calvete (Instituto de Biomedicina de Valencia, Spain) for technical support and
suggestions. B.S.C., A.H.S. and V.J.F.F. are senior investigators of CNPq/Brazil.
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Received on February 2, 2002, accepted on June 5, 2002.
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Article
  • Sep 1970
Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
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Quantitation of boar spermadhesins in accessory sex gland fluids and on the surface of epididymal, ejaculated and capacitated spermatozoa
Article
  • Jun 1994
  • Biochim Biophys Acta
Spermadhesins are multifunctional proteins involved in boar sperm capacitation and gamete recognition. Using anti-AWN antibodies, we have followed the fate of spermadhesin AWN along the maturation and capacitation stages of boar spermatozoa. In addition, the amount of spermadhesins AQN-1, AQN-2, and AQN-3 relative to that of AWN was determined by amino acid analysis after reverse-phase HPLC isolation. Our data show that AWN-1 is the only spermadhesin on the surface of epididymal sperm and that a large amount of AQN-1, AQN-2, AQN-3, AWN-1 and AWN-2 become coated on ejaculated spermatozoa. The number of spermadhesin molecules on ejaculated sperm (12-60 x 10(6)/spermatozoa) is sufficient to form a many-molecules-thick coat over the sperm head. However, 50-75% of the AQN-1, AQN-2, and AQN-3 population, and around 90% of coated AWN (1 + 2) are released from ejaculated sperm during capacitation. This indicates that a large subpopulation of each boar spermadhesin is loosely associated to the sperm surface and may function as decapacitation factors. The remaining spermadhesin molecules, which are tightly bound to the sperm head's surface may play a role as either positive capacitation factors and/or in gamete recognition and binding.
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Immunocytochemical localization of spermadhesin AWN in the porcine genital tract
Article
  • Nov 1995
Boar spermadhesin (AWN) is a 14-kDa multifunctional protein, attached to the surface of the spermatozoa and involved in sperm capacitation and zona pellucida binding. The cellular origin of AWN was previously unknown. Moreover, the region of the male genital tract in which AWN becomes attached to the surface of spermatozoa was also uncertain. By using monospecific polyclonal antibodies against AWN, the immunohistochemical distribution pattern of AWN epitopes has been investigated in tissue sections of the porcine male genital tract. Our study has revealed that AWN is synthesized in the rete testis and in the epithelium of the seminal vesicles. The latter are also the major contributors of seminal plasma AWN. In addition, immunoblotting analysis has shown that AWN is present on epididymal spermatozoa. Our results indicate that the cellular origin of spermadhesins is species-specific. The attachment of AWN to epididymal spermatozoa is probably important in developing the capacity for fertilization.
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Crystallization and preliminary X-ray diffraction analysis of boar seminal plasma spermadhesin PSP-I/PSP-II, a heterodimer of two CUB domains
Article
  • Apr 1996
Boar spermadhesin PSP-I/PSP-II (M(r) 29 000-30 000), a non-covalent heterodimer of two CUB domains, was crystallized in two crystal forms. Complete diffraction data sets for hexagonal (space group P6(1,5)22) and trigonal (space group P3(1,2)21) crystals have been collected up to 2.9 and 2.5 angstrom resolution, respectively. Cell constants of the hexagonal and trigonal crystal forms are a=b=87.2 angstrom, c=152.4 angstrom, and a=b=96.2 angstrom, c=70.8 angstrom, respectively. The calculated packing parameters (Vm) are 2.8 and 3.2 angstrom(3)/DA for the hexagonal and trigonal crystal forms, respectively, indicating that, in both cases, the asymmetric unit is constituted by one PSP-I/PSP-II heterodimer. This paper reports the first crystals of a protein built up by a CUB domain architecture.
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Elation and characterization of heparin- and phosphorylcholine-binding proteins of boar and stallion seminal plasma. Primary structure of porcine pB1
Article
  • May 1997
In the bovine, seminal plasma heparin-binding proteins bind to sperm lipids containing the phosphorylcholine group and mediate the capacitating effects of heparin-like glycosaminoglycans during sperm residence in the female genital tract. We report the characterization of heparin- and phosphorylcholine-binding proteins of stallion and boar seminal plasma. orse eminal lasma proteins HSP-1 and HSP-2, and boar protein pB1, belong to the same family as the bull heparin- and phosphorylcholine-binding proteins BSP-A1/2, BSP-A3, and BSP-30K. We have determined the amino acid sequence and posttranslational modifications of boar glycoprotein pB1. It contains 105 amino acids arranged into a mosaic structure consisting of a N-terminal 18-residue O-glycosylated polypeptide followed by two tandemly organized 40–45-residue fibronectin type II domains. pB1 displays 60–65% amino acid sequence similarity with its equine and bovine homologues. However, in their respective seminal plasmas, the BSP and the HSP proteins associate into 90–150-kDa oligomeric complexes, whereas pB1 forms a 35–40-kDa complex with spermadhesin AQN-1. In addition, pB1 appears to be identical to the recently described leukocyte adhesion regulator of porcine seminal fluid pAIF-1. Our results tie in with the hypothesis that homologous proteins from different mammalian species may display distinct biological activities, which may be related to species-specific aspects of sperm physiology.
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Binding of mannose-6-phosphate and heparin by boar seminal plasma PSP-II, a member of the spermadhesin protein family
Article
  • Aug 1998
PSP-I/PSP-II, a heterodimer of glycosylated spermadhesins, is the major component of boar seminal plasma. Similarly to other spermadhesins, the PSP-II subunit is a lectin which displays heparin- and zona pellucida glycoprotein-binding activities. We have investigated the ligand binding capabilities of the heterodimer and the isolated subunits using several polysaccharides, glycoproteins, and phospholipids. PSP-II binds the sulfated polysaccharides heparin and fucoidan in a dose-dependent and seemingly-specific manner. In addition, PSP-II binds oligosaccharides containing exposed mannose-6-phosphate monoester groups and the binding is selectively inhibited by mannose-6-phosphate and glucose-6-phosphate. Inhibition experiments indicate that binding of PSP-II to sulfated polysaccharides and mannose-6-phosphate-containing oligosaccharides involves distinct but possibly overlapping binding sites. Heterodimer formation with PSP-I abolishes both the heparin and the mannose-6-phosphate binding capabilities, suggesting that the corresponding sites may be located at the dimer interface. Using the crystal structure of PSP-I/PSP-II heterodimer as a template, we have explored possible binding sites which satisfy the observed binding characteristics. In the proposed models, PSP-II Arg43 appears to play a pivotal role in both heparin- and mannose-6-phosphate-complexation as well as in heterodimer formation.
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  • Jan 1997
  • 179-186
  • M Reinert
  • J J Calvete
  • L Sanz
  • E Topfer-Petersen
Reinert, M., Calvete J.J., Sanz, L. and Topfer-Petersen, E. (1997). Andrology, 29, 179-186.
  • Jan 1997
  • 201-206
  • J J Calvete
  • M Raida
  • M Gentzel
  • C Urbanke
  • L Sanz
  • E Topfer-Petersen
Calvete, J.J., Raida, M., Gentzel, M., Urbanke, C., Sanz, L. and Topfer-Petersen, E. (1997). FEBS Letters, 407, 201-206. [4]