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Eine wichtige Eigenschaft der Plasmamembran eukaryotischer Zellen ist die stabile transversale Asymmetrie der Phospholipide. Sie wird energieabhängig durch die Aktivität einer Aminophospholipidtranslokase aufrechterhalten und gilt als wichtige Voraussetzung für die Homöostasis der Zellen. Die Plasmamembran einiger Säugerzellen weist zudem laterale Lipiddomänen auf, denen eine wesentliche Bedeutung bei der Signaltransduktion zugeschrieben wird. Während der Genese durchlaufen die Membranen der Säugerspermien intensive Veränderungen. Um die Bedeutung der Phospholipidasymmetrie für die Funktion der Spermien zu untersuchen, wurde die Lokalisation und Dynamik von Phosphatidylserin in der Zell- und Akrosommembran von Eberspermien im Verlauf von Kapazitation und Akrosomreaktion betrachtet. Unter Ausnutzung der selektiven, kalziumabhängigen Bindung von AnnexinV an endogenes Phosphatidylserin konnte dessen Lokalisation an morphologisch differenzierten Zellen verfolgt werden. Eine Markierung der Zellen mit NBD-markierten Phospholipidanaloga lieferte zudem Informationen zur Dynamik der Phospholipide in der Plasmamembran. Die Differenzierung der Zellen erfolgte entweder am Durchflusszytometer oder fluoreszenz- bzw. elektronenmikroskopisch. Die Ergebnisse der vorliegenden Arbeit weisen sowohl auf eine transversale als auch laterale Ungleichverteilung der Lipide in der Zell- und Akrosommembran während der Genese der Spermien hin. Neben der stabilen transversalen Phospholipidasymmetrie der Plasmamembran konnte erstmals eine zytoplasmatische Lokalisation von Phosphatidylserin auf der äußeren Akrosommembran nachgewiesen werden. Somit akkumulieren die beiden einander zugewandten zytoplasmatischen Monolayer von Plasmamembran und äußerer Akrosommembran Phosphatidylserin. Kapazitationsbedingt kommt es zu einer engen Wechselwirkung zwischen Plasmamembran und äußerer Akrosommembran. Die Ausbildung lateraler Membrandomänen, in denen Phosphatidylserin zytoplasmatisch akkumuliert, wird als Vo, One of the essential qualities of cell membranes in Eucaryotae is a stable transverse phospholipid asymmetry. It is regulated and maintained by ATP-dependent action of an aminophospholipid translocase and is a major prerequisite for cell homeostasis. The plasma membranes of several mammalian cells show moreover lateral lipid domains, which are imputed to play a significant role in signal transduction. The membranes of mammalian spermatozoa undergo significant changes during genesis. The localisation and dynamics of phosphatidylserine in the cell as well as acrosome membranes of boar sperm cells was studied during capacitation and acrosome reaction to assess the relevance of lipid asymmetry for sperm function. The localisation of endogenous phosphatidylserine in morphologically differentiated cells was followed using the selective calcium depending binding of annexinV. Information on the transverse dynamics of phospholipids in the plasma membrane was obtained by labelling the cells with a NBD-phospholipid analogues. The morphological status of the cells was assessed by flow cytometry, fluorescence and electron microscopy. The results of this study indicate both a transversal and lateral inhomogenous distribution of lipids in the cell membrane as well as in the outer acrosome membrane during sperm genesis. The plasma membrane of boar sperm shows a stable transversal lipid asymmetry characterised by an accumulation of phosphatidylserine in the cytoplasmic monolayer. Moreover a cytoplasmic localisation of phosphatidylserine on the outer acrosome membrane could be detected for the first time. Therefore the two facing cytoplasmic leaflets of the outer acrosome and cell membrane contain phosphatidylserine. Applying microscopy substantiated the hypothesis that there are close interactions between the cell membrane and the outer layer of the acrosome membrane because of capacitation. The cytoplasmic accumulation of phosphatidylserine in lateral lipid domains is probably esse

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 P61,522) and trigonal (space group P31,221) crystals have been collected up to 2.9 and 2.5 Å resolution, respectively. Cell constants of the hexagonal and trigonal crystal forms are a = b = 87.2 Å, c = 152.4 Å, and a = b = 96.2 Å, c = 70.8 Å, respectively. The calculated packing parameters (V(m)) are 2.8 and 3.2 Å3/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.

We report the complete amino acid sequence of HSP-1, a major protein isolated from stallion seminal plasma or acid extracts of ejaculated spermatozoa. The protein consists of 121 amino acids organized in two types of homologous repeats arranged in the pattern AA'BB'. Each of the 13-15-residue A-type repeats contains two O-linked oligosaccharide chains. The B-type repeats span 44-47 amino acids each, are not glycosylated, and have the consensus pattern of the gelatin-binding fibronectin type-II module. This domain also occurs in the major bovine seminal plasma heparin-binding proteins PDC-109 (BSP-A1/A2) and BSP-A3. However, unlike the bovine proteins which bind quantitatively to a heparin-Sepharose column, stallion HSP-1 was recovered in both the flow-through and the heparin-bound fractions. Structural analysis showed that the two HSP-1 forms contain identical polypeptide chains which are differently glycosylated. Moreover, size-exclusion chromatography showed that heparin-bound HSP-1 associates with HSP-2, another major seminal plasma protein, into a 90 kDa product, whereas the non-heparin-bound glycoform of HSP-1 is eluted as a monomeric (14 kDa) protein. This suggests that glycosylation may have an indirect effect on the heparin-binding ability of HSP-1 through modulation of its aggregation state. On the other hand, both glycoforms of HSP-1 displayed gelatin-binding activity, indicating that the molecular determinants for binding heparin and gelatin are different.

Spermadhesin PSP-II was isolated from the non-heparin-binding fraction of boar seminal plasma; its disulphide bridge pattern, and the location of a single N-glycosylation site were established. PSP-II forms a heterodimer with specific N-glycoforms of PSP-I. Although both subunits possess heparin-binding capability, the PSP-I/PSP-II complex does not. The heterodimer contains binding sites for zona pellucida glycoproteins and soybean trypsin inhibitor located in the PSP-II subunit. However, the PSP-I/PSP-II heterodimer binds only loosely to the sperm surface and is easily removed during in vitro capacitation, suggesting that the zona pellucida binding activity may not be relevant for gamete interaction. Our results show that dimerization of spermadhesins PSP-I and PSP-II markedly affects their binding capabilities. © 1995.

Spermadhesin AWN-1 (14 kDa) belongs to a recently described family of boar sperm surface-associated proteins. AWN-1 is a multifunctional protein which possesses heparin-, serine proteinase inhibitor-, and zona pellucida glycoprotein-binding capability. Therefore it has been implicated in sperm capacitation and sperm-oocyte attachment. Here, we report the characterization of 22-25 kDa isoforms of AWN-1 isolated by heparin-affinity chromatography, which fail to bind to zona pellucida glycoproteins or serine proteinase inhibitors. Our results show that the structure of the high and low molecular mass AWN-1 forms differ in that the former is N-glycosylated at Asp50 and truncated at the C-terminus. The inability of the glycosylated AWN-1 molecules to bind ligands is due solely to the presence of the oligosaccharide moieties, however. This indicates that glycosylation of AWN-1 may modulate its ligand-binding capabilities. On the other hand, the effect of glycosylation on ligand-binding suggests that both the zona pellucida- and the serine proteinase inhibitor binding domain(s) may be located around the glycosylation point. © 1993.