Characterization of human sperm plasma membrane: glycolipids and polypeptides

The plasma membranes from ejaculated human spermatozoa were removed by nitrogen cavitation (600 PSI for 10 min) and isolated by centrifugation followed by a discontinuous sucrose density gradient centrifugation. Glycolipid analysis of the plasma membrane revealed a three-fold enrichment in gangliosides: GM3 and GD1a/GD1b and neutral glycolipids: globoside and sulfatide as compared to that of whole human sperm. Two dimensional electrophoresis of human sperm plasma membranes revealed about 75 polypeptides. Several of these polypeptides were similar in migration and in display of shape and color to that found in boar sperm plasma membranes.     

Human blood group A and H glycolipids in porcine plasma. Evidence for acquisition of the erythrocyte antigens from plasma

Human blood group A- and H-antigenic glycosphingolipids were isolated from pooled porcine plasma. The structures of the A-active hexa- and H-active pentaglycosylceramides of lactoseries (type 1 sugar chain) were the same as those in porcine erythrocytes. These results endorse biochemically the previous observations that the A and H antigens on porcine erythrocytes are taken up from plasma.     

Characterization of porcine plasma fibronectin and its fragmentation by porcine liver cathepsin B

Fibronectin was isolated from porcine plasma by affinity chromatography with gelatin-linked Sepharose 4B. Porcine fibronectin had a chemical composition similar to those of human and other fibronectins and reacted with antiserum raised against human fibronectin. It showed hemagglutination activity with trypsin-treated rabbit erythrocytes, though the activity was far less than that of human fibronectin. Porcine plasma fibronectin consisted of two subunit chains of about 230,000-daltons linked by disulfide bonds(s). Limited proteolysis of this protein with porcine liver cathepsin B yielded five major fragments which were investigated by affinity chromatography with gelatin- and heparin-linked Sepharose 4B. One fragment (Mr = 50,000) was bound to gelatin but not to heparin, while the remaining four were bound to heparin but not to gelatin, suggesting that plasma fibronectin takes a discrete domain structure with respect to interaction with these two macromolecules. The three larger heparin-binding fragments, Mr = 175,000, 150,000, and 130,000 were eluted with different concentrations of a mixture of NaCl and urea from the heparin-column, suggesting that they have different interactions with heparin, the 130,000-dalton fragment being the one with the strongest interaction. After reduction with 2-mercaptoethanol, the 175,000-dalton fragment was converted to the 150,000-dalton region fragment, which, together with the unchanged 150,000-dalton fragment, appeared to be equivalent in amount to the 130,000-dalton fragment. This finding suggests that the 150,000- and 130,000-dalton fragments may have originated from different subunit chains. Since the 175,000-dalton fragment was not produced by cathepsin B digestion of fibronectin which had been treated with plasmin, it was concluded that the 175,000-dalton fragment contained interchain disulfide bond(s) which had linked the native subunit chains. These results suggest that porcine plasma fibronectin has non-identical subunit chains composed of domains which differ in interaction with heparin and in susceptibility to cathepsin B.     

Characterization of major glycolipids in bovine erythrocyte membrane

Several neutral glycolipids and gangliosides were isolated from bovine erythrocyte stroma. Their structures were determined by partial acid hydrolysis, methylation analysis, periodate oxidation and CrO3 oxidation. Two major neutral glycolipids were characterized as lactosylceramide and galactosyl(alpha1--3)galactosyl(beta1--4)N-acetylglucosaminyl(beta1--3)galactosyl(beta1--4)glucosyl(beta1--1)ceramide. Two major gangliosides were N-glycolylneuraminosyl(2--3)galactosyl(beta1--4)glucosyl(beta1--1)ceramide and N-glycolylneuraminosyl(2--3)galactosyl(beta1--4)N-acetylglucosaminyl(beta1--3)galactosyl(beta1--4)glucosyl(beta1--1)ceramide. Minor glycolipids were glucosyl- and galactosylceramide, glucosamine-containing tri- and tetraglycosylceramide, glucosamine-containing disialosylhexaglycosylceramide, and gangliosides containing N-acetylneuraminic acid. The ceramide moiety of each glycolipid contained perdominantly d18:1 sphingosine, and normal fatty acids of C16:0, C22:0, C24:0, and C24:1.     

Structural characterization of neutral and acidic glycolipids from Thermus thermophilus HB8

The structural characterization of glycolipids from Thermus thermophilus HB8 was performed in this study. Two neutral and one acidic glycolipids were extracted and purified by the modified TLC-blotting method, after which their chemical structures were determined by chemical composition analysis, mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. The structure of one of the neutral glycolipids, NGL-A, was Galp(α1-6)GlcpNacyl(β1-2)Glcp(α1-)acyl(2)Gro, and the other, NGL-C, was Galf(β1-2)Galp(α1-6)GlcpNacyl(β1-2)Glcp(α1-)acyl(2)Gro. The structure of NGL-C was identical to that reported previously [Oshima, M. and Ariga, T. (1976) FEBS Lett. 64, 440]. Both neutral glycolipids shared a common structural unit found in the Thermus species. The acyl groups found in NGL-A and NGL-C, iso-type pentadecanoxy and heptadecanoxy fatty acid, were also the same as those found in this species. In contrast, the acidic glycolipid, AGL-B, possessed the structure of N-(((GlcpNAc(α1-)acyl(2)Gro)P-2)GroA)alkylamine. The alkyl group in AGL-B was an iso-type heptadecanyl, suggesting that the iso-type structure of the long alkyl chain is responsible for the thermal stability of the bacteria.     

Confirmation of minor components of less polar neutral and acidic glycolipids in monkey brain tissue

Crude glycolipids, prepared without alkali treatment in advance, were separated into neutral and acidic glycolipids by DEAE-Sephadex A-25 (acetate form) column chromatography. Each glycolipid was further fractionated by a Silica gel 60-column chromatography. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with delayed ion extraction (DE MALDI-TOF MS) of the intact glycolipid fractions, the less polar neutral glycolipids were found to contain alkali-labile ester cerebrosides and Galb-1-Diradylglycerols, whereas the less polar acidic glycolipids were found to contain alkali-labile ester sulfatide, HSO(3)-3Gal-1-Diradylglycerols, and novel alkali-stable plasmalo-sulfatides and ester or plasmalo HSO(3)-3Galb-1-Diradylglycerols as minor components of glycolipids in monkey brain tissue.In conclusion, minor components of less polar neutral and acidic glycolipids in monkey brain tissue were confirmed as ester cerebrosides, Galb-1-Diradylglycerols, ester sulfatides, HSO(3)-3Galb-1-Diradylglycerols, and novel plasmalo-sulfatides and ester or plasmalo HSO(3)-3Galb-1-Diradylglycerols by DE MALDI-TOF MS.     

Complex formation of 70-kDa heat shock protein with acidic glycolipids and phospholipids

A new property of a heat-inducible heat shock protein (Hsp) 70.1 that it forms a complex with acidic lipids was first demonstrated. Based on the behaviors of the complexes on the native PAGE, the acidic lipid/Hsp70.1 complexes are categorized into two groups. The first group is the sulfatide-induced large-sized complex, which stays on the gel top on the native PAGE. Only the N-terminal ATPase domain is responsible for the complex formation. The second group is the ganglioside-induced complex, which is diffused in the resolution gel on the native PAGE. Both the N-terminal ATPase and the C-terminal peptide-binding domains are involved in the complex formation. No complex is formed by neutral glyco- and phospholipids. The complex formation with the acidic glyco- and phospholipids implicates the various functions of Hsp70 on the membrane surfaces.     

Characterization of porcine plasma protein-based films as affected by pretreatment and cross-linking agents

The effects of pretreatment and cross-linking agents on properties of porcine plasma protein-based film were investigated. Based on sodium dodecyl sulfate polyacrylamide gel electrophoresis and solubility study, porcine plasma protein-based film was stabilized mainly by hydrogen bond, while film with pretreatment (pH 10 and heated for 30 min) contained hydrogen bond and hydrophobic interaction. The incorporation of glyoxal or caffeic acid resulted in the increase in protein cross-links stabilized by both disulfide and non-disulfide covalent bonds. Nevertheless, the prior oxygenation had no marked impact on the property of film added with caffeic acid. α-Chymotrypsin was more effective than pepsin in hydrolysis of films. Greater thermal stability with an increase in the melting point was observed in film incorporated with caffeic acid, indicating a greater degree of cross-linking. The coincidental increase in initial temperature of film degradation was noticeable. The FTIR spectra revealed the intermolecular interaction between protein molecules as indicated by the shift of amide-I peak.     

Characterization of capacitation, cryoinjury, and the role of seminal plasma in porcine sperm

Capacitation is a biochemical pathway sperm must undergo to be able to fertilize an oocyte, whereas cryoinjury is cryopreservation-induced biophysical damage which renders sperm immediately capable of fertilization. Similarities between capacitation and cryoinjury have not been fully elucidated. The present study attempted to characterize both processes, including the role of seminal plasma (SP). Merocyanine-540 staining detected an increase (P < 0.01) in plasma membrane disorder from 60.5% in in vitro capacitated sperm to 91.4% in cryopreserved sperm, with no effect of SP. After cryopreservation, 42.8% of sperm displayed phosphatidylserine on the outer leaflet compared to 13.6% of in vitro capacitated sperm (P < 0.01), as assessed by annexin-V staining (SP decreased phosphatidylserine inversion in both populations). Lipid raft-associated glycolipid GM₁ movement increased throughout the entire sperm membrane in cryopreserved sperm, although SP did not affect lipid raft movement in these sperm. Cryopreserved and in vitro capacitated sperm had a similar intensity of tyrosine phosphorylation (although SP reduced this intensity). In in vitro capacitated sperm, 67.5% underwent an ionophore induced acrosome reaction with 91.3% reacting in cryopreserved sperm. In both cases, SP reduced (P < 0.01) the percentage of acrosome-reacted sperm to 1.0 and 7.8%, respectively. Cryopreservation appeared to damage sperm, resulting in marked increases in membrane disorder, cholesterol efflux, and percent of capacitated sperm. In both capacitated and cryoinjured sperm, the addition of SP appeared to attenuate some of these events.     

Characterization of bioactive glycolipids from Scytonema julianum (cyanobacteria)

Cyanobacteria serve as a rich source of novel bioactive metabolites. Few studies on glycolipids reported them as having specific biological activities. In this study, total lipids of Scytonema julianum, a filamentous cyanobacterium isolated from a Greek cave, were separated into neutral and phospho- and glycolipids, and the latter were further fractionated by high-performance liquid chromatography (HPLC). Each glycolipid fraction was tested in vitro for its ability to inhibit platelet-activating factor (PAF)- and thrombin-induced washed rabbit platelet aggregation and/or to cause platelet aggregation. The structures of the most active fractions were elucidated by biological assays, by chemical determinations and identified by electrospray mass spectrometry. One fraction was a potent inhibitor of PAF-induced platelet aggregation. Structural studies of this fraction indicated the existence of a phosphoglyco-analog of acyl-sphingosine. Two fractions causing platelet aggregation were detected and identified as phosphoglycolipids. The first one was identified as a phosphoglyco-analog of acyl-acetylated sphingosine and the second one as a glyco-analog of phosphatidylglycerol. The presence of the above bioactive compounds demonstrates new types of lipids in cyanobacteria in regard to the structure and biological activity. In addition, the identified bioactive lipids may contribute to the allergic character of cyanobacteria.     

Characterization of porcine adrenocortical lysosomes

Porcine adrenocortical lysosomes were characterized by differential centrifugation, acid hydrolase contents, latency of cathepsin D, release of bound acid hydrolases in soluble form, and isopycnic density gradient centrifugation. Cathepsins D and B, beta-N-acetylglucosaminidase, beta-galactosidase and arylsulphatase were found exclusively in the lysosomes, while alpha-mannosidase and beta-glucuronidase were in both the lysosomal and microsomal fractions. The activity of cathepsin D was remarkably high, amounting to more than 6 times that in porcine liver and to more than 10 times that in liver of Sprague-Dawley rats in terms of units per g wet tissue. Porcine adrenocortical lysosomes showed a modal isopycnic density value of 1.155, but mitochondria a value of 1.145. The validity of these values was studied by investigating the possibilities of agglutination of organelles, damage to lysosomal membranes, disruption of mitochondria due to the hydrostatic pressure and by applying the same procedures of isopycnic centrifugation to hog and rat livers. After these validity tests, porcine adrenocortical lysosomes were concluded to be unique in their strikingly high content of cathepsin D as well as in their low modal isopycnic density which is very close to that of porcine adrenocortical mitochondria.     

The presence of -mannosidic linkage in acidic glycopeptide from porcine thyroglobulin

The mannose residue in (Man)₁ (GlcNAc)₂-Asn obtained by a Smith degradation of the acidic glycopeptide from porcine thyroglobulin was found to be insusceptible to α-mannosidase. This residue was hydrolyzed, however, by purified β-mannosidase. After β-mannosidase treatment, the resulting (GlcNAc)₂-Asn was compared with synthetic glycosyl-asparagine derivatives. From these experiments, the core structure of the acidic glycopeptide was proposed to be β-Man-(1 → 3 or 4)-β-GlcNAc-(1 → 4)-GlcNAc-Asn.