Water-soluble glycoproteins from Cannabis sativa (South Africa)

The water-soluble glycoproteins obtained from Cannabis leaves of plants grown from South African seeds have been further studied. Treatment of the glycoprotein fractions with NaOH in the presence of NaBH₄, resulted in a significant decrease in the serine content and a corresponding increase in alanine. The carbohydrate side chains released contained the sugar alcohol, galactitol. By treatment of the glycoprotein fractions with NaOH in the presence of Na₂SO₃, and subsequent acid hydrolysis, cysteic acid was formed. These data indicate that carbohydrate and protein are connected via serine-O-galactoside linkages. Further investigation of the structure of the carbohydrate part of the glycoproteins was carried out by methylation analysis, Smith-degradation and enzyme incubation. The present glycoprotein material of plants grown from South African seeds is similar to the material previously investigated, but in contrast to the latter, it is devoid of hexosamine.     

The amino acid sequence of Cannabis sativa cytochrome-c

The amino acid sequence of hemp (Cannabis sativa L.) cytochrome-c was determined using 0.7 μmol of protein. Analysis of chymotryptic and tryptic peptides by the dansyl Edman method showed that the molecule consisted of 111 residues and was homologous with other mitochondrial plant cytochromes-c.     

Cell wall glycoproteins from Chlamydomonas reinhardii are sulphated

Abstract Both the two major structural cell wall glycoproteins and the soluble excreted glycoproteins of Chlamydomonas reinhardii Levine WT II/32 contain low levels (approx. 1–4%) of sugar O-sulphate esters, asymmetrically distributed within the molecules. Preliminary characterization of their structure is described through [³⁵S] sulphate labelling experiments. The function of the sulphated glycoproteins is discussed in terms of their structural role and their water retaining properties.     

Methylation analysis of cell wall glycoproteins and glycopeptides from Chlamydomonas reinhardii

Cell wall glycoproteins from Chlamydomonas reinhardii and the glycopeptides produced by the action of thermolysin were subjected to standard methylation analysis. GC-MS of the methylated alditol acetates revealed short oligosaccharides some of which show branching. O-glycosidically linked galactofuranosyl residues are present. The asymmetric distribution of the major O-glycosidic linkages is also reported.     

A GLYCOPROTEIN NONCOVALENTLY ASSOCIATED WITH CELL‐WALL POLYSACCHARIDE OF THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA)1

The cells of the red microalga Porphyridium sp. (UTEX 637) are encapsulated in a cell wall of a negatively charged mucilaginous polysaccharide complex composed of 10 different sugars, sulfate, and proteins. In this work, we studied the proteins associated with the cell‐wall polysaccharide. A number of noncovalently associated proteins were resolved by SDS‐PAGE, but no covalently bound proteins were detected. The most prominent protein detected was a 66‐kDa glycoprotein consisting of a polypeptide of approximately 58 kDa and a glycan moiety of approximately 8 kDa containing N‐linked terminal mannose. In size‐exclusion chromatography, the 66‐kDa protein was coeluted with the polysaccharide and could be separated from the polysaccharide only after denaturation of the protein, indicating that the 66‐kDa protein was tightly bound to the polysaccharide. Western blot analysis revealed that the 66‐kDa protein was specific to Porphyridium sp. and P. cruentum, because it was not detected in the other species of red microalgae examined. Indirect immunofluorescence assay confirmed the location of the protein in the algal cell wall. The sequence of cDNA clone encoding the 66‐kDa glycoprotein, detected in our in‐house expressed sequence tag database of Porphyridium sp., revealed that this is a novel protein with no similarity to any protein in the public domain databases and our in‐house expressed sequence tag database of the red microalga Rhodella reticulata. The 66‐kDa protein bound polysaccharides from red algae but not from those of other origins tested. Possible roles of the 66‐kDa protein in the biosynthesis of the polysaccharide are discussed.     

Water-soluble polysaccharides of Opuntia ficus-indica cv “Burbank's Spineless”

Carbohydrate-containing polymers have been extracted with water from the fleshy, lobed stems of Opuntia ficus-indica cv “Burbank's Spineless”. By ion exchange chromatography, the material was separated into one neutral and two acidic fractions. Each fraction was separated in two by gel filtration. The neutral fractions consisted of two glucans and a glycoprotein, containing arabinose and galactose. All four acidic fractions contained galacturonic acid, arabinose, rhamnose, galactose and xylose in different proportions. The cell wall structure of O. ficus-indica is discussed.     

盐胁迫下枸杞叶片细胞表面糖蛋白的变化

利用细胞化学的方法,对经过不同浓度盐胁迫的枸杞细胞表面糖蛋白的动态变化进行了标记定位电镜观察。枸杞盐胁迫处理后发现,盐胁迫过程中枸杞细胞表面糖蛋白层明显增厚、致密。随着胁迫时间的延长则变薄、脱落,游离到细胞间隙中。细胞璧糖蛋白的积累是枸杞对盐胁迫反应的重要生化机制,在枸杞的耐盐生理活动中起着重要作用。     

Detecting and minimizing glycosidase activities that can hydrolyze sugars from cell culture-produced glycoproteins

Heterogeneity of cell culture-produced glycoproteins often results from the presence or absence of a few sugars found on the terminus of glycoprotein oligosaccharides. Variability in bioprocess factors can potentially lead to variability in this oligosaccharide heterogeneity (1). Although stochastic events in the intracellular biosynthetic process have long been recognized as a cause of oligosaccharide heterogeneity (2), more recent data has demonstrated that extracellular degradation by glycosidases can also contribute to oligosaccharide heterogeneity (3,4). The purpose of this chapter is to introduce the concept and consequence of glycosidase degradation, to discuss methods for evaluating whether glycosidase degradation is significant for a particular process, and to provide some potential remedies to alleviate undesirable degradation.     

An epitope of rice threonine- and hydroxyproline-rich glycoprotein is common to cell wall and hydrophobic plasma-membrane glycoproteins

A monoclonal antibody, LM1, has been derived that has a high affinity for an epitope of hydroxyproline-rich glycoproteins (HRGPs). In suspension-cultured rice (Oryza sativa L.) cells the epitope is carried by three major proteins with different biochemical properties. The most abundant is the 95-kDa extracellular rice extensin, a threonine- and hydroxyproline-rich glycoprotein (THRGP) occurring in the cell wall and secreted into the medium. This THRGP can be selectively oxidatively cross-linked in the presence of hydrogen peroxide and an endogenous peroxidase with the result that it does not enter a protein gel. A second polypeptide with the LM1 epitope (180 kDa), also occurring in the suspension-cultured cells and medium, is not oxidatively cross-linked. Three further polypeptides (52, 65 and 110 kDa) with the characteristics of hydrophobic proteins of the plasma-membrane also carry the LM1 epitope as determined by immuno-blotting of detergent/aqueous partitions of a plasma-membrane preparation and immuno-fluorescence studies with rice protoplasts. At the rice root apex the LM1 epitope is carried by four glycoproteins and is developmentally regulated. The major locations of the epitope are at the surface of cells associated with the developing protoxylem and metaxylem in the stele, the longitudinal radial walls of epidermal cells and a sheath-like structure at the surface of the root apex.Abbreviations AGP arabinogalactan protein - ELISA enzyme-linked immunosorbent assay - HRGP hydroxyproline-rich glycoprotein - THRGP threonine- and hydroxyproline-rich glycoprotein This work was supported by The Leverhulme Trust. We also acknowledge support from The Royal Society and thank Prof. L.A. Staehelin for the carrot extensin, N. Stacey for the rice cell culture and Dr. J. Keen for protein sequencing.     

Folding of viral envelope glycoproteins in the endoplasmic reticulum

Viral glycoproteins fold and oligomerize in the endoplasmic reticulum of the host cell. They employ the cellular machinery and receive assistance from cellular folding factors. During the folding process, they are retained in the compartment and their structural quality is checked by the quality control system of the endoplasmic reticulum. A special characteristic that distinguishes viral fusion proteins from most cellular proteins is the extensive conformational change they undergo during fusion of the viral and cellular membrane. Many viral proteins fold in conjunction with and dependent on a viral partner protein, sometimes even synthesized from the same mRNA. Relevant for folding is that viral glycoproteins from the same or related virus families may consist of overlapping sets of domain modules. The consequences of these features for viral protein folding are at the heart of this review.     

Isolation of glycoproteins of parainfluenza I (Sendai) by Helix pomatia Lectin column

Glycoproteins of Sendai virus were successfully isolated on a column of Helix pomatia Lectin-Sepharose 6MB following solubilization with Nonidet P-40. This technique can be used as a preparative step for the study of viral glycoproteins.