Paul H. Atkinson

Rhodopsin's protein and carbohydrate structure: Selected aspects

A topographic model for rhodopsin has been constructed based upon evaluation of rhodopsins sequence by a secondary structure prediction algorithm as well as chemical and enzymatic modification of rhodopsin in the membrane [Hargrave et al. (1983) Biophys. Struct. Mech. 9, 235-244]. The non-uniform distribution of several amino acids in the primary structure and within the topographic model is discussed. The seven predicted helices were evaluated and each helix was found to have one surface which is much more hydrophobic than the other. Stereoscopic views of a three dimensional model with a functional color-coding scheme incorporating these features are presented. The amino acid sequence of rhodopsin has been compared to other proteins in the Dayhoff Protein Data Bank. No obvious relationship to any other protein sequenced was found. High resolution proton magnetic resonance spectroscopy was used to reinvestigate the structure and relative proportions of rhodopsins major and minor oligosaccharide chains. One major (Man3GlcNAc3) and two minor (Man4GlcNAc3 and Man5GlcNAc3) were observed.

Cell-surface glycopeptides: Growth-dependent changes in the carbohydrate-peptide linkage region☆

Abstract Glycopeptides were isolated from the surface of human diploid cells maintained strictly in the growing, and non-growing, state by daily feeding at optimum pH. By use of extensive pronase and endoglycosidase (Muramatsu, 1971) digestion we reduced surface glycopeptides to a fragment containing only a few amino acids and sugar moieties. We report a growth-dependent alteration near the carbohydrate-peptide linkage region. The heterogeneous glycopeptide fragments representing this region have an average molecular weight of approximately 800. Partial characterization of these fragments show that they contain fucose in α-linkage, hexosamine and aspartic acid.

Major carbohydrate structures at five glycosylation sites on murine IgM determined by high resolution 1H-NMR spectroscopy☆

Mouse myeloma immunoglobulin IgM heavy chains were cleaved with cyanogen bromide into nine peptide fragments, four of which contain asparagine-linked glycosylation. Three glycopeptides contain a single site, including Asn 171, 402, and 563 in the intact heavy chain. Another glycopeptide contains two sites at Asn 332 and 364. The carbohydrate containing fragments were treated with Pronase and fractionated by elution through Bio-Gel P-6. The major glycopeptides from each site were analyzed by 500 MHz 1H-NMR and the carbohydrate compositions determined by gas-liquid chromatography. The oligosaccharide located at Asn 171 is a biantennary complex and is highly sialylated. The amount of sialic acid varies, and some oligosaccharides contain alpha 1,3-galactose linked to the terminal beta 1,4-galactose. The oligosaccharides at Asn 332, Asn 364, an Asn 402 are all triantennary and are nearly completely sialylated on two branches and partially sialylated on the triantennary branch linked beta 1,4 to the core mannose. The latter is sialylated about 40% of the time for all three glycosylation sites. The major oligosaccharide located at Asn 563 is of the high mannose type. The 1H-NMR determination of structures at Asn 563 suggests that the high mannose oligosaccharide contains only three mannose residues.

Alterations in Glycoproteins of the Cell Surface

It has been a widely held view over the last several years that the surface membrane of mammalian cells can initiate growth regulatory events when presented with specific external stimuli. This idea has stemmed mainly from the effect of various substances which are presumed not to enter the target cell and elicit a specific response in the cell.

Incorporation of fucose into HeLa cell plasma membranes during the cell cycle.

Abstract The metabolism of HeLa cell plasma membranes during the cell cycle was studied by following the incorporation of radioactive precursor l -[ 3 H]fucose into plasma membranes of synchronized cells. Maximal incorporation of the radioactive precursor was observed in late S phase of the cell cycle. This discrete period of increased incorporation of precursor into the plasma membranes implies the existence of a distinct control mechanism which may relate cell surface phenomena to the cell cycle.

1H NMR spectroscopy of carbohydrates from the G glycoprotein of vesicular stomatitis virus grown in parental and Lec4 Chinese hamster ovary cells

Carbohydrate moieties derived from the G glycoprotein of Vesicular Stomatitis Virus (VSV) grown in parental Chinese hamster ovary (CHO) cells and the glycosylation mutant Lec4 have been analyzed by high-field 1H NMR spectroscopy. The major glycopeptides of CHO/VSV and Lec4/VSV were purified by their ability to bind to concanavalin A-Sepharose. The carbohydrates in this fraction are of the biantennary, complex type with heterogeneity in the presence of alpha(2,3)-linked sialic acid and alpha (1,6)-linked fucose residues. A minor CHO/VSV glycopeptide fraction, which does not bind to concanavalin A-Sepharose but which binds to pea lectin-agarose, was also investigated by 1H NMR spectroscopy. These carbohydrates are complex moieties which appear to contain N-acetylglucosamine in beta(1,6) linkage. Their spectral properties are most similar to those of a triantennary complex oligosaccharide containing a 2, 6-disubstituted mannose alpha (1,6) residue. Carbohydrates of this type are not found among the glycopeptides of VSV grown in the Lec4 CHO glycosylation mutant.

Separation of high-mannose isomers from yeast and mammalian sources using high-pH anion-exchange chromatography

The biosynthesis of oligosaccharides N-linked at AsnXxxThr(Ser) sequons of glycoproteins is initiated by the co-translational transfer of a Glc,Man,GlcNAc, from dolichyl pyrophosphate’. The three glucose residues are rapidly removed by two glucosidases, one a( I+ 2)and the other a( 1 +3)-specific’. These first steps are identical in yeast and in man, and likely to be the same in plants’? however, the subsequent removal and addition of the peripheral mannoses are very different. The array of oligo-mannosyl structures which eventually occupy each glycosylation site are a function, in part, of the concerted action of mannosidases and mannosyl transferases on the archetypal Man,GlcNAc, structure’. These enzymes often display specificity for not only the type of residue and anomeric linkage, but also for its branch location. For example, a yeast mannosidase removes a single (l-+2)-x-linked mannose from a specific branch of the Man,GlcNAcz oligosaccharide4,‘. A yeast (1+6)-a-mannosyl transferase places a single mannose on one specific branch which is essential for elongation reaction?. Trimming by at least three mammalian mannosidases accounts for the heterogeneity of high-mannose structures at individual glycosylation sites (discussed in ref. 7). Thus, chromatographic methods which can separate not only by size and ring substitutions (linkage), but also according to branch isomerism, are required to understand the structural glycobiology of iv-linked oligosaccharide biosynthesis. High-pH anion-exchange chromatography (h.p.a.e.c.) has been shown to separate many oligosaccharide isomers (both neutral and charged) which differ only in a single linkagex~‘4~‘7). H owever, certain isomers were difficult to separate using

Fractionation of ovalbumin glycopeptide AC-C by high-pressure liquid chromatography Determination of structure by 1H-NMR spectroscopy

We describe for the first time a method for the separation of intact ovalbumin glycopeptides by high-pressure liquid chromatography (HPLC). The separation was achieved using two reverse-phase columns connected in series and eluting with an isocratic solvent system at acid pH containing 1-hexane sulfonate. Ovalbumin glycopeptide fraction AC-C has been separated into at least four distinct glycopeptides. High resolution 1H-NMR spectroscopy has confirmed the reported structure of the two major species. We also extend our structural studies to the two other glycopeptides and establish the structure of a previously unreported ovalbumin glycopeptide, Man3GalGlcNAcAsn.

Chapter 8 HeLa Cell Plasma Membranes

Publisher Summary This chapter describes a method for isolation of HeLa cell plasma membranes. The method has the advantage of being rapid (with practice, a product can be obtained in about 45 minutes) and does not require the use of uncommon equipment. Some observations on the disposition of fucose-containing glycoproteins in HeLa cells are also included, together with preliminary data on the kinetics of their arrival in the plasma membrane. The general design of the method described for the preparation of HeLa cell plasma membranes is based on a series of rate zonal sedimentations which serves to differentially separate cellular components of disrupted cells. The plasma membrane product of various methods has one of three main morphologies, namely: fragments, ghosts, and vesicles. The first methodology utilizes gentle disruption of the liver cells in hypotonic solution followed by the isolation of plasma membranes as large fragments possibly “bile fronts” as identified in the phase contrast and electron microscope. This approach became the basis for many modifications in the isolation of plasma membranes from liver tissues of various mammals. A second and basically different methodology does not preserve the ghost structure, but rather disrupts the cells vigorously in isotonic buffer, converting the entire surface membrane into vesicles. In the process of homogenization, membrane vesicles from other sources also become mixed with the surface membranes.

Determination of the structure of ovalbumin glycopeptide AC-B by 1H nuclear magnetic resonance spectroscopy at 500 MHz.

Three unique, unmodified ovalbumin glycopeptides were separated to homogeneity by high-pressure liquid chromatography. The nuclear magnetic resonance data, at 500 MHz, confirmed the structure of two of the three species and for the first time established the presence of a Man8GlcNAc2Asn glycopeptide in ovalbumin. This compound was a single homogeneous isomeric form out of three possible compounds expected as processing intermediates.