Functions of red cell surface proteins

The external membrane of the red cell contains numerous proteins that either cross the lipid bilayer one or more times or are anchored to it through a lipid tail. Many of these proteins express blood group activity. The functions of some of these proteins are known; in others their function can only be surmised from the protein structure or from limited experimental evidence. They are loosely divided into four categories based on their functions: membrane transporters; adhesion molecules and receptors; enzymes; and structural proteins that link the membrane with the membrane skeleton. Some of the proteins carry out more than one of these functions. Some proteins may complete their major functions during erythropoiesis or may only be important under adverse physiological conditions. Furthermore, some might be evolutionary relics and may no longer have significant functions. Polymorphisms or rare changes in red cell surface proteins are often responsible for blood groups. The biological significance of these polymorphisms or the selective pressures responsible for their stability within populations are mostly not known, although exploitation of the proteins by pathogenic micro-organisms has probably played a major role.     

Translational diffusion in phospholipid monolayers measured by fluorescence microphotolysis.

A method is described that eliminates surface flow in monolayers at the air-water interface and makes possible diffusion measurements by fluorescence microphotolysis ("photobleaching"). In contrast to previous studies that did not account for surface flow, lipid probe diffusion has been found to be similar in densely packed monolayers and in related bilayers. Furthermore, it seems that lipid diffusion is based on the same molecular mechanism in monolayers, bilayers, and potentially also cell membranes. In monolayers of L-alpha-dilauroylphosphatidylcholine (Lau2-PtdCho) the translational diffusion coefficient D of the fluorescent lipid probe N-4-nitrobenzo-2-oxa-1,3 diazole egg phosphatidylethanolamine decreased from 110 microns2/s at a surface pressure II = 1 mN/m to 15 microns2/s at II = 38 mN/m (T = 21-22 degrees C). Data could be fitted by the "free volume model." In monolayers of L-alpha-dipalmitoylphosphatidylcholine (Pam2-PtdCho) D decreased by greater than 3 orders of magnitude upon increasing II at constant temperature, thus indicating a fluid-to-crystalline phase transition. In Lau2-PtdCho/Pam2-PtdCho monolayers phase separation has been visualized in the fluorescence microscope and the effect on D measured. These results suggest that monolayers are a promising model system for studying the molecular mobility of lipids and other cell membrane components.     

Positive selection on protein-length in the evolution of a primate sperm ion channel

Positive Darwinian selection on advantageous point substitutions has been demonstrated in many genes. We here provide empirical evidence, for the first time, that positive selection can also act on insertion/deletion (indel) substitutions in the evolution of a protein. CATSPER1 is a voltage-gated calcium channel found exclusively in the plasma membrane of the mammalian sperm tail and it is essential for sperm motility. We determined the DNA sequences of the first exon of the CATSPER1 gene from 15 primates, which encodes the intracellular N terminus region of approximately equal to 400 aa. These sequences exhibit an excessively high frequency of indels. However, all indels have lengths that are multiples of 3 nt (3n indels) and do not disrupt the ORF. The number of indel substitutions per site per year in CATSPER1 is five to eight times the corresponding rates calculated from two large-scale primate genomic comparisons, which represent the neutral rate of indel substitutions. Moreover, CATSPER1 indels are considerably longer than neutral indels. These observations strongly suggest that positive selection has been promoting the fixation of indel mutations in CATSPER1 exon 1. It has been shown in certain ion channels that the length of the N terminus region affects the rate of channel inactivation. This finding suggests that the selection detected may be related to the regulation of the CATSPER1 channel, which can affect sperm motility, an important determinant in sperm competition.     

Phospholipid bilayer surface configuration probed quantitatively by (31)P field-cycling NMR

(31)P relaxation of the diester phosphate of phospholipids in unilamellar vesicles has been studied from 0.004 to 11.7 T. Relaxation at very low fields, below 0.1 T, shows a rate increase that reflects a residual dipolar interaction with neighboring protons, probably dominated by the glycerol C3 protons. This interaction is not fully averaged by faster motion such as rotational diffusion perpendicular to the membrane surface. The remaining dipolar interaction, modulated by overall rotational diffusion of the vesicle and lateral diffusion of the lipid molecules, is responsible for the very low-field relaxation. These measurements yield a good estimate of the time-average angle between the membrane surface and the vector connecting the phosphorus to the glycerol C3 protons, based on the classic theory by Woessner. Dynamic information is also obtained. Implications for solid-state NMR and other studies are discussed.     

日本血吸虫精子的超微结构

目的：了解日本血吸虫正常精子的超微结构。方法：半超薄切片定位雄虫睾丸和含有成熟精子的雌虫输卵管，常规透射电镜制样并观察。结果：日本血吸虫精子由头、尾两部分组成，头部呈长卵圆形，平均长6．2μm，宽1．4μm，无顶体构造，前端钝圆，后端尖细，质膜下有1圈纵行的微管，核1个，前端有少量线粒体，尾部鞭毛1根，在中、后段主体部分鞭毛轴丝外周为9组二联管，中央为一团弥散的电子致密物质，呈9×2＋《1》型；但在过渡区鞭毛轴丝中央无电子致密物质，呈9×2＋0型。结论：日本血吸虫精子超微结构与其他血吸虫相似，具有同源性，但明显区别于复殖目大多数吸虫的构造。     

Ultrastructural observation of spermatozoa and fertilization in Schistosoma japonicum

The ultrastructure of the sperm and the process of fertilization are described in Schistosoma japonicum. The sperm of S. japonicum has an elongated head and a single tail. The head measures 6.2 x 1.4 microm in average size. No acrosome is present. A mass of mitochondria locates in front of the nucleus. A layer of about 100-120 peripheral microtubules is parallel with the long axis of the head under plasma membrane. The nucleus is dense with some electron-lucent patches. The tail is a single flagellum with unique axoneme, which originates from a centriole. The structure of axoneme includes two types: 9 x 2 + in the main part of the flagellum, and 9 x 2 + 0 near the end of the flagellum. The sperm ultrastructure of S. japonicum is similar to that of other schistosomes, apart from the fact that two types of configuration coexisted in the same axoneme, and there is no striated root found in S. japonicum. The sperm differs distinctly from other Digenea. The aberrant ultrastructure of S. japonicum reflects that its evolution is far away from other genera in Digenea. Fertilization occurs at the posterior part of oviduct, in which region the oviduct wall lacks lamellae. Some cortical granules (CG) fuse with plasma membrane, and discharge their content on the surface of the fertilized ovum. The other CGs break down or degenerate in the cytoplasm. By the secondary mature division, the secondary oocyte finally divides to form a female pronucleus. During this period a male pronucleus also forms. The female and male pronucleus approach each other, come into contact in the central region and finally fuse to form a zygote. The function of CGs is discussed.     

Lateral diffusion of surface immunoglobulin, Thy-1 antigen, and a lipid probe in lymphocyte plasma membranes

Fluorescence photobleaching recovery was used to measure the lateral diffusion coefficient and mobile fraction of surface immunoglobulin (sIg), Thy-1 antigen, and a lipid probe in the plasma membrane of mouse lymphocytes. The lipid probe (3,3'-dioctadecylindocarbocyanine) had a mean (+/-SD) diffusion coefficient of (1.7 +/- 0.3) x 10(-8) cm(2)/sec, with essentially all of the probe mobile in the membrane. We detected little or no effect on the diffusion of this probe due to the presence of microvilli. Its diffusion was slightly restricted in capped regions. No differences in lipid probe mobility were detected between T and B cells. Fifty to 90% of the detectable sIg and Thy-1 antigen was free to move in the plane of the membrane with diffusion coefficients of approximately 3 x 10(-10) cm(2)/sec; the remainder was immobile. Crosslinking of sIg with anti-Ig antibodies (in the presence of azide to inhibit capping) completely immobilized sIg at high concentrations but failed to do so at low concentrations. Thy-1 antigen could not be immobilized with an IgG rabbit anti-mouse brain reagent without an additional layer of crosslinking antibody. In parallel labelings (in the absence of azide), capping of sIg and Thy-1 antigen was observed only under crosslinking conditions sufficient to immobilize the membrane antigen. Sodium azide, colchicine, and cytochalasin B had no measurable effect on lipid probe, sIg, or Thy-1 diffusion.     

Erythrosomes: large proteoliposomes derived from crosslinked human erythrocyte cytoskeletons and exogenous lipid.

Large (3-micrometers diameter) mechanically stable proteoliposomes (erythrosomes) were prepared in good yield by coating crosslinked erythrocyte cytoskeletons with phosphatidylcholine. The erythrosomes consist of the polypeptides designated band 1, 2, 3, 4.1 + 4.2, and 5 (less than 4% of the endogenous lipid) and enough added lipid to form a bilayer coating the surface. Electron microscopy shows only the large proteoliposomes in sealed preparations. The trapping of bovine serum albumin, mannitol, sucrose, glucose, cytosine arabinoside, and sodium in the erythrosomes was demonstrated, yielding an apparent volume of up to 100 liters/mol of phospholipid. This preparation possesses an effective diffusion barrier to glucose, sucrose, and sodium ion with half-equilibration times of 34, 29, and 170 hr, respectively. The results of the present study suggest that erythrosomes may be useful for membrane transport protein reconstitution and encapsulation systems.     

Conservation of honey bee (Apis mellifera) sperm phospholipids during storage in the bee queen — A TLC/MALDI–TOF MS study

The honey bee (Apis mellifera) is characterized by a high degree of phenotypic plasticity of senescence-related processes, and has therefore become a model organism of gerontological research. Sperm of honey bee drones can remain fertile for several years within the storage organ of queens. The reason for this longevity is unknown, but the suppression of lipid peroxidation seems to play a decisive role. Here, we examined the questions of whether spermatheca- and in vitro-stored honey bee sperm are indeed resistant to lipid peroxidation, and whether the nature of sperm lipids could explain this resistance. The lipid composition of bee sperm was determined by matrix-assisted laser desorption and ionization time-of-flight (MALDI–TOF) mass spectrometry (MS) combined with thin-layer chromatography (TLC). The positive ion mass spectra of drone sperm lipids are dominated by two glycerophosphocholine (GPC) species, although small amounts of sphingomyelins (SM) and glycerophosphoethanolamines (GPE) are also detectable after TLC. Alkyl/acyl and alkenyl/acyl compounds of GPC, and alkyl/acyl as well as diacyl compounds of GPE were detected containing oleyl, oleoyl, palmityl and palmitoyl as the most abundant residues. Assignments of all compounds have been additionally verified by enzymatic digestion and exposition to HCl. During incubation of sperm in the presence of air, characteristic lipid oxidation products such as lysophosphatidylcholine (LPC) appear. Inside the spermatheca, however, sperm lipids are obviously protected from oxidation and their composition does not change, even if they are stored over years. Our data support the view that the membrane composition of honey bee sperm could help to explain the extraordinary longevity of these cells.     

On a Theory for the Passive Transport of Solute through Semipermeable Membranes

It has been shown that thin-film dialysis can be performed in such a way that the limiting rate is the rate of entry of the solute into the membrane from the high-concentration side. The rate of diffusion, therefore, reflects the probability of a molecule entering the pores on the surface and does not depend on the resistance to diffusion offered by the internal structure of the membrane. The good correlation, so generally found, of the order of escape times of given solutes with the order of free diffusion rates is thus explained. The data from stretching experiments with wet cellophane, in which the pore structure is distorted, are also explained.     

Involvement of trypsin-like activity in binding of mouse spermatozoa to zonae pellucidae.

Work from a number of laboratories has shown that fertilization is blocked in the presence of protease inhibitors, although the specific site of inhibition has not been identified. The present experiments were designed to discriminate between sperm binding to zonae pellucidae as opposed to sperm penetration through zonae, so as to assess the effect of protease inhibitors on these two distinct events. Exposure of capacitated mouse spermatozoa to a variety of protease inhibitors directed against trypsin blocked sperm binding to zonae in a concentration-dependent manner. A chymotrypsin-directed inhibitor was not capable of blocking sperm binding to zonae. The trypsin inhibitors did not affect sperm penetration though zonae nor gamete membrane fusion if the sperm had established a firm association with the zona surface before addition of the inhibitors. Previous incubation of zona-intact eggs with the inhibitors did not lead to a reduction in sperm binding, indicating that the activity affected by the inhibitors is borne by spermatozoa. Interaction between spermatozoa and the zona surface appeared to be the specific locus of inhibition; sperm binding to zona-free eggs (i.e., binding to the egg plasma membrane) was unaltered by the trypsin inhibitors. These results suggest a reevaluation of the function of proteases in fertilization focusing on their role in initial sperm contact with the zona pellucida.     

A novel lipid-conjugated marker for the direct labeling of the schistosomulum membrane

Introduction of the fluorescent group lissamine rhodamine B into the surface of schistosomula of S. mansoni was achieved by brief incubation of the worms with liposomes carrying the lipid bound fluorophore in their bilayers. The liposomes were made of egg lecithin (PC) and lissamine-phosphatidylethanolamine (lissamine-PE). The lissamine groups could be directly detected on the parasite membrane by observation of the single worms under a fluorescent microscope. The fluorescent marker was employed to measure the lateral diffusion coefficient (D) of lipids in the schistosomular membrane and as a surface marker during the isolation of the schistosomula surface membrane.     

Recent aspects of mammalian fertilization research

Mammalian fertilization has been the subject of intensified research in recent times. Application of recombinant DNA, transgenic and gene targeting technology, in particular, to issues in mammalian fertilization has revolutionized the field. Here, we present some of the latest results coming from application of these and other technologies to four aspects of mammalian fertilization: 1. formation of the egg zona pellucida (ZP) during oocyte growth; 2. species-specific binding of sperm to the egg zona pellucida; 3. induction of the sperm acrosome reaction (AR) by the egg zona pellucida 4. binding of sperm to and fusion with egg plasma membrane. In virtually every instance, new information and new insights have come from relatively recent investigations.     

Slaved diffusion in phospholipid bilayers

The translational diffusion of phospholipids in supported fluid bilayers splits into two populations when polyelectrolytes adsorb at incomplete surface coverage. Spatially resolved measurements using fluorescence correlation spectroscopy show that a slow mode, whose magnitude scales inversely with the degree of polymerization of the adsorbate, coexists with a fast mode characteristic of naked lipid diffusion. Inner and outer leaflets of the bilayer are affected nearly equally. Mobility may vary from spot to spot on the membrane surface, despite the lipid composition being the same. This work offers a mechanism to explain how nanosized domains with reduced mobility arise in lipid membranes.     

Fusion of liposomes with mitochondrial inner membranes

A procedure is outlined for the fusion of mixed phospholipid liposomes (small unilamellar vesicles) with the mitochondrial inner membrane, which enriches the membrane lipid bilayer 30-700% in a controlled fashion. Fusion was initiated by manipulation of the pH of a mixture of freshly sonicated liposomes and the functional inner membrane/matrix fraction of rat liver mitochondria. During the pH fusion procedure, liposomes became closely apposed with and sequestered by the inner membranes as revealed by freeze-fracture electron microscopy. After the pH fusion procedure, a number of ultrastructural, compositional, and functional characteristics were found to be proportionally related: the membrane surface area increased; the lateral density distribution of intramembrane particles (integral proteins) in the plane of the membrane decreased whereas the particles remained random; the membrane became more buoyant; the ratio of membrane lipid phosphorus to total membrane protein increased; the ratio of membrane lipid phosphorus to heme a of cytochrome c oxidase increased; and the rate of electron transfer between some interacting membrane oxidoreduction proteins decreased. These data reveal that liposomal phospholipid was incorporated into the membrane bilayer (not simply adsorbed to the membrane surface) and that integral membrane proteins diffused freely into the laterally expanding bilayer. Furthermore, the data suggest that the rate of electron transfer may be limited by the rate of lateral diffusion of oxidoreduction components in the bilayer of the mitochondrial inner membrane.     

Affinity of talin-1 for the β3-integrin cytosolic domain is modulated by its phospholipid bilayer environment

Binding of the talin-1 FERM (4.1/ezrin/radixin/moesin) domain to the β3 cytosolic tail causes activation of the integrin αIIbβ3. The FERM domain also binds to acidic phospholipids. Although much is known about the interaction of talin-1 with integrins and lipids, the relative contribution of each interaction to integrin regulation and possible synergy between them remain to be clarified. Here, we examined the thermodynamic interplay between FERM domain binding to phospholipid bilayers and to its binding sites in the β3 tail. We found that although both the F0F1 and F2F3 subdomains of the talin-1 FERM domain bind acidic bilayers, the full-length FERM domain binds with an affinity similar to F2F3, indicating that F0F1 contributes little to the overall interaction. When free in solution, the β3 tail has weak affinity for the FERM domain. However, appending the tail to acidic phospholipids increased its affinity for the FERM domain by three orders of magnitude. Nonetheless, the affinity of the FERM for the appended tail was similar to its affinity for binding to bilayers alone. Thus, talin-1 binding to the β3 tail is a ternary interaction dominated by a favorable surface interaction with phospholipid bilayers and set by lipid composition. Nonetheless, interactions between the FERM domain, the β3 tail, and lipid bilayers are not optimized for a high-affinity synergistic interaction, even at the membrane surface. Instead, the interactions appear to be tuned in such a way that the equilibrium between inactive and active integrin conformations can be readily regulated.     

The Thy-1 antigen exhibits rapid lateral diffusion in the plasma membrane of rodent lymphoid cells and fibroblasts.

Thy-1 is a plasma membrane protein, but its primary structure lacks the typical membrane-spanning sequence. Recent studies revealed that a glycophospholipid is covalently bound to the carboxyl terminus, suggesting that the protein is integrated into the plasma membrane by this lipid moiety. Lateral diffusion of Thy-1 was measured in mouse thymocytes, lymphoma cells, and fibroblasts by the fluorescence recovery after photobleaching technique. Thy-1 was labeled with rhodamine-conjugated anti-Thy-1 monoclonal antibodies. Diffusion coefficients of 2-4 X 10(-9) cm2/sec were obtained for the antigen-antibody complex in all the cell types. About 50% of the Thy-1 was mobile. The diffusion coefficient for the mobile fraction of Thy-1 is considerably larger than the diffusion coefficients of many other plasma membrane proteins. Rather, the diffusion coefficient of Thy-1 is similar to those of lipid analogs embedded in the same membrane, providing strong support for the suggested lipid anchoring of this antigen.     

Progesterone acts at the plasma membrane of human sperm.

There has been increasing interest in the relationship between rapid effects of steroids and steroid-plasma membrane interaction. This laboratory has previously reported that progesterone increases human sperm cytosolic free calcium ([Ca2+]i) and thereby initiates the human sperm acrosome reaction (AR) in less than 1 min. Herein, to test whether progesterone acts at the sperm plasma membrane, progesterone 3-(O-carboxymethyl)oxime: bovine serum albumin (BSA) conjugate (free of unconjugated progesterone) was added to capacitated human sperm. Fura-2 assays were used to detect less than 1 min changes in [Ca2+]i, and indirect immunofluorescence was used to assay the AR occurring 1 min after stimulus addition. The conjugate increased [Ca2+]i and the AR (though less than did unconjugated progesterone). Enzyme immunoassays demonstrated that the concentrations of unconjugated progesterone in conjugate-treated sperm suspensions did not increase over those of control suspensions. Since the progesterone: BSA conjugate presumably does not cross the sperm plasma membrane, progesterone must act at that membrane to increase [Ca2+]i and the AR.