Glycoproteins and glycolipids of oxyntic cell microsomes II. Glycopeptides and glycolipids

Glycosylated compounds associated with the carbohydrate-rich tubular membrane system of the oxyntic cell were investigated. Two glycopeptide fractions, designated Peaks A and B, were isolated from pronase digests of bullfrog oxyntic cell microsomes. Molecular sieve chromatography and cellulose acetate electrophoresis revealed that, although somewhat heterogeneous, each peak was composed primarily of glycopeptides with similar molecular weights and net charge densities. Peak B glycopeptides had a mean molecular weight of about 6000 and contained 70% of the recovered carbohydrate in the following molar ratios: hexose, 1.00; $\text{N-}\text{acetylhexosamine}$, 0.71; fucose, 0.61; sialic acids, <0.03. Peak a glycopeptides were considerably larger (molecular weight approx. 100 000) and contained carbohydrates in molar ratios similar to those of Peak B. In both peaks galactose and $\text{N-}\text{acetylglucosamine}$, respectively, were the predominant hexose and amino sugar isomers.The glycolipid content of bullfrog oxyntic cell microsomes was assessed by qualitative and quantitative thin-layer chromatography. The most abundant glycolipids were monoglucosylceramides (0.098 mole/mole phospholipid) and monogalactosylceramides (0.046 mole/mole phospholipid). Small quantities of sulfatides and gangliosides were also present.A compilation of available data regarding the chemical composition of the microsomes revealed that these membranes resemble plasma membranes in having high molar ratios of cholesterol to phospholipid (approx. 1.0) and large quantities of carbohydrate (225 μg/mg protein). The possible significance of these compositional features in protecting the oxyntic cell is discussed.     

Synthesis of glycolipids

The chemical syntheses of naturally occurring glycolipids derived from sphingosine bases and glycerol derivatives, and the syntheses of polyisoprenoid lipid intermediates and other miscellaneous glycolipids recorded up to the end of 1977 are reviewed.     

Bilayer Edges Catalyze Supported Lipid Bilayer Formation

Supported lipid bilayers (SLB) are important for the study of membrane-based phenomena and as coatings for biosensors. Nevertheless, there is a fundamental lack of understanding of the process by which they form from vesicles in solution. We report insights into the mechanism of SLB formation by vesicle adsorption using temperature-controlled time-resolved fluorescence microscopy at low vesicle concentrations. First, lipid accumulates on the surface at a constant rate up to ∼0.8 of SLB coverage. Then, as patches of SLB nucleate and spread, the rate of accumulation increases. At a coverage of ∼1.5 × SLB, excess vesicles desorb as SLB patches rapidly coalesce into a continuous SLB. Variable surface fluorescence immediately before SLB patch formation argues against the existence of a critical vesicle density necessary for rupture. The accelerating rate of accumulation and the widespread, abrupt loss of vesicles coincide with the emergence and disappearance of patch edges. We conclude that SLB edges enhance vesicle adhesion to the surface and induce vesicle rupture, thus playing a key role in the formation of continuous SLB.     

Sulfated glycolipids and cell adhesion

The adhesive glycoproteins laminin, thrombospondin, and von Willebrand factor bind specifically and with high affinity to sulfatides, and it is this binding that probably accounts for their ability to agglutinate glutaraldehyde-fixed erythrocytes. The three proteins differ, however, in the inhibition of their binding to sulfatides by sulfated polysaccharides. Fucoidan strongly inhibits binding of both laminin and thrombospondin, but not of von Willebrand factor, suggesting the involvement of laminin or thrombospondin, or other unknown sulfatide-binding proteins in specific cell interactions that are also inhibited by fucoidan. Thrombospondin adsorbed on plastic promotes the attachment and spreading of some melanoma cells. Interestingly, fucoidan and an antibody against the sulfatide-binding domain of thrombospondin selectively inhibit spreading but not attachment to thrombospondin-coated surfaces. Sulfatides, but not neutral glycolipids or gangliosides, when adsorbed on plastic also promote attachment and spreading of some cultured cell lines. Direct adhesion of melanoma cells requires high densities of adsorbed sulfatide. In the presence of laminin, however, specific adhesion of some cell types to sulfatide is strongly stimulated and requires only low densities of adsorbed lipid, suggesting that laminin is mediating adhesion by crosslinking receptors on the cell surface to sulfatide adsorbed on the plastic. Although thrombospondin also binds to sulfatides and to melanoma cells, it does not enhance but rather inhibits direct and laminin-dependent melanoma cell adhesion to sulfatide, presumably because it is unable to bind simultaneously to ligands on opposing surfaces. Thus, sulfated glycolipids can participate in both laminin- and thrombospondin-mediated cell adhesion, but their mechanisms of interaction are different.     

Phospholipids and glycolipids of sterol-requiring Mycoplasma

The phospholipids of Mycoplasma hominis type 2 strain 07 are composed almost entirely of phosphatidyl glycerol. Traces of other glycerophospholipids may exist. No glycolipids are found. The phospholipids of Mycoplasma sp. avian strain J are composed of diphosphatidyl glycerol, which predominates in older cultures, a monoacyl glycerophosphoryl glycerophosphate, which may serve as a precursor of diphosphatidyl glycerol, and phosphatidyl glycerophosphate. This organism also contains cholesteryl glucoside and an unidentified glycolipid which appears to be similar to a monoglucosyl diglyceride. No turnover or radioisotope labeling of the phospholipids occurs during metabolism. This lack of turnover during growth is indicative of a structural role for these glycerophospholipids. A concomitant decrease of monoacyl glycerophosphoryl glycerophosphate and increase of diphosphatidyl glycerol occurs during growth.     

Polymyxin B Loosens Lipopolysaccharide Bilayer but Stiffens Phospholipid Bilayer

Multidrug-resistant Gram-negative bacteria have increased the prevalence of a variety of serious diseases in modern times. Polymyxins are used as the last-line therapeutic options for the treatment of infections. However, the mechanism of action of polymyxins remains in dispute. In this work, we used a coarse-grained molecular dynamics simulation to investigate the mechanism of the cationic antimicrobial peptide polymyxin B (PmB) interacting with both the inner and outer membrane models of bacteria. Our results show that the binding of PmB disturbs the outer membrane by displacing the counterions, decreasing the orientation order of the lipopolysaccharide tail, and creating more lipopolysaccharide packing defects. Upon binding onto the inner membrane, in contrast to the traditional killing mechanism that antimicrobial peptides usually use to induce holes in the membrane, PmBs do not permeabilize the inner membrane but stiffen it by filling up the lipid packing defect, increasing the lipid tail order and the membrane bending rigidity as well as restricting the lipid diffusion. PmBs also mediate intermembrane contact and adhesion. These joint effects suggest that PmBs deprive the biological activity of Gram-negative bacteria by sterilizing the cell.     

Bilayer membranes and transporter models

Significant advances in the understanding of the mechanisms of artificial transporters appeared in the past year which provide insights into natural transporters and suggest directions for regulation of transport. Parallel developments in the control of bilayer formation and molecular recognition by synthetic lipids have led to simple mimics of ‘whole cell’ types of processes.     

Model membranes bearing glycolipids and glycoproteins

The forces that hold cell membrane components together are non-covalent and thermodynamically favoured in aqueous media. Hence virtually any glycolipid or membrane glycoprotein might be expected to be incorporable into lipid bilayer membranes and this expectation has been borne out. In addition methods have been developed for linking lipid fragments to species that would not otherwise be expected to associate with bilayers. Techniques that have been successfully used to generate bilayer structures bearing glycolipids and glycoproteins include hydration of films dried down from non-aqueous solutions of the components, detergent removal from aqueous component solutions, exogenous addition to preformed membranes, and various organic solvent injection or reverse phase approaches. Bilayer association of glycolipids and membrane glycoproteins, with preservation of specific receptor function, seem easy to achieve — in fact difficult not to achieve. Optimization of receptor function to accurately mimic that of cell membranes and efficient preservation of functions such as transport or second messenger activation, are typically more demanding, although still feasible. A systematic approach can give considerable insight into the processes involved via identification of minimal necessary factors. Unfortunately, the actual relative arrangement of components, so critical to subtleties of glycolipid and glycoprotein function, remains almost totally unknown for lack of morphological information in the size range of individual macromolecules. The latter problem has come to be the most critical limitation to many studies.     

ABH-blood-group antigens and glycolipids of human saliva

The nature of ABH-blood-group antigens in saliva was investigated. Human saliva was examined serologically for ABH-blood-group activity in its native form and after various treatments. The activity of the native form persisted in the delipidated samples, but was entirely lost after alkaline degradation. The lipid portion of saliva was completely inactive in the ABH hemagglutination inhibition system. The same results were obtained when purified glycolipid fraction of saliva was used instead of whole lipid extract. Neither alkaline treatment nor excessive amounts of salivary lipids effected antigenic activity of A-active glycosphingolipids of hog gastric mucosa admixed to saliva samples before alkaline degradation and/or in presence of large amounts of salivary lipids. The isolated glycolipid fractions contained at least eight glycolipids, each of which was composed of glucose, glyceryl ethers and fatty acids and differed from others with respect to number of glucose residues. Sphingosine and sugar residues involved in formation of ABH antigenic determinants were not detected. These findings together with data on stomach secretion [1,2] led us to the conclusion that ABH-blood-group antigens of saliva are exclusively of glycoprotein nature.