A modeling framework for the study of protein glycosylation

The key step in the asparagine-linked glycosylation of secretory proteins is the transfer of oligosaccharide from a dolichol precursor to the polypeptide at an Asp-X-Ser/Thr (NXS/T) consensus sequence. It is often the case, both in cultured cells and in vivo, that this reaction does not occur for every molecule of a given protein. Thus, the cell may create two protein populations, one bearing and one lacking oligosaccharide, for each potential glycosylation site. We present a structured kinetic modeling framework of the initial glycosylation event based on a balance of available glycosylation sites through the region of endoplasmic reticulum lumen proximal to the membrane. Oligosaccharyltransferase, a multimeric protein complex, catalyzes the sugar transfer. This enzyme is integral to the endoplasmic reticulum membrane, and it is thought to act cotranslationally. The nascent polypeptide may also fold in such a way as to prevent glycosylation from occurring. The net result is a potentially complex spatial and temporal relationship among translation, glycosylation, and other cotranslational events. Model results predict how fractional glycosylation site occupancy may depend on protein synthesis rate, oligosaccharyldolichol availability, and mRNA elongation rate. Although we are currently unable to quantitatively compare predicted to experimentally obtained fractional site occupancy, we are able to determine qualitative trends which may be confirmed experimentally. (c) 1996 John Wiley & Sons, Inc.     

High throughput screening for compounds that alter muscle cell glycosylation identifies new role for N-glycans in regulating sarcolemmal protein abundance and laminin binding

Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function.     

The role of protein glycosylation in the control of cellular N-sialyltransferase activity

Protein glycosylation, which is a key post-translational event, is catalysed by the glycosyltransferase family of enzymes. There is an increasing body of evidence to suggest that these enzymes may themselves be glycosylated, possibly as an autocatalytic event. Using a novel in vitro system, we have investigated the role of enzyme glycosylation in sialyltransferase catalytic activity. The enzyme activity is glycosylation dependent, with the penultimate galactose residue on complex N-linked oligosaccharides playing a pivotal role. These results serve to underline the complexity of the glycosylation process.     

A novel HPLC-UV-MS method for quantitative analysis of protein glycosylation

Monoclonal antibody samples derived from transgenic plants (plantibodies) may often contain significant amounts of aglycosylated variants. Because glycosylated and non-/de-glycosylated proteins exhibit different functional and pharmacokinetic properties, accurate measurement of non- and de-glycosylated glycoprotein abundances is important. Glycosylation of plant-derived glycoproteins presents specific challenges. Here we describe a novel method to accurately measure relative and absolute amounts of non-glycosylated, de-glycosylated, and total glycosylated protein using an HPLC-UV-MS methodology. Additionally, these results were compared with glycopeptide profiling by MALDI MS. Our studies demonstrated that the quantitative aspect of HPLC-UV method was superior to MALDI MS profiling, which significantly overestimated the relative amounts of aglycosylated species in the isolated glycopeptide fractions.     

Sorting out glycosylation enzymes in the Golgi apparatus

The study of glycosylation and glycosylation enzymes has been instrumental for the advancement of Cell Biology. After Neutra and Leblond showed that the Golgi apparatus is the main site of glycosylation, elucidation of oligosaccharide structures by Baenziger and Kornfeld and subsequent mapping of glycosylation enzymes followed. This enabled development of an in vitro transport assay by Rothman and co-workers using glycosylation to monitor intra Golgi transport which, complemented by yeast genetics by Schekman and co-workers, provided much of the fundamental insights and key components of the secretory pathway that we today take for granted. Glycobiology continues to play a key role in Cell Biology and here, we look at the use of glycosylation enzymes to elucidate intra Golgi transport.     

A new role for laminins as modulators of protein toxicity in Caenorhabditis elegans

Protein misfolding is a common theme in aging and several age-related diseases such as Alzheimer's and Parkinson's disease. The processes involved in the development of these diseases are many and complex. Here, we show that components of the basement membrane (BM), particularly laminin, affect protein integrity of the muscle cells they support. We knocked down gene expression of epi-1, a laminin α-chain, and found that this resulted in increased proteotoxicity in different Caenorhabditis elegans transgenic models, expressing aggregating proteins in the body wall muscle. The effect could partially be rescued by decreased insulin-like signaling, known to slow the aging process and the onset of various age-related diseases. Our data points to an underlying molecular mechanism involving proteasomal degradation and HSP-16 chaperone activity. Furthermore, epi-1-depleted animals had altered synaptic function and displayed hypersensitivity to both levamisole and aldicarb, an acetylcholine receptor agonist and an acetylcholinesterase inhibitor, respectively. Our results implicate the BM as an extracellular modulator of protein homeostasis in the adjacent muscle cells. This is in agreement with previous research showing that imbalance in neuromuscular signaling disturbs protein homeostasis in the postsynaptic cell. In our study, proteotoxicity may indeed be mediated by the neuromuscular junction which is part of the BM, where laminins are present in high concentration, ensuring the proper microenvironment for neuromuscular signaling. Laminins are evolutionarily conserved, and thus the BM may play a much more causal role in protein misfolding diseases than currently recognized.     

An obligatory role of protein glycosylation in the life cycle of yeast cells

Two water-soluble carbodiimides, differing in molecular dimensions, have been used to characterize the cytochrome c binding site of bovine heart cytochrome c oxidase. Several polypeptide components of the enzyme contain acidic residues which are modified by these reagents. Carboxyl groups present in subunit II, VII and polypeptide c, are protected from modification when cytochrome c, equimolar to oxidase, is added and they can cross-link to the substrate once activated by the carbodiimide. Comparison of the modification patterns suggest that the most reactive residues are located on subunit II and VII, the former being also more exposed. The data obtained indicate that even though subunit II plays the major role in binding cytochrome c, at least two other lower Mr polypeptides contribute to the cytochrome c binding domain.     

Life is sweet! A novel role for N-glycans in Drosophila lifespan

N-glycans are post-translational modifications in which the sugar chain is covalently linked to protein by a GlcNAcβ1-N-asparagine linkage. Drosophila melanogaster and other invertebrates, but not vertebrates, synthesize large amounts of "paucimannose" N-glycans that contain only three or four mannose residues. The enzyme UDP-GlcNAc:α3-D-mannoside β1,2-N-acetylglucosaminyltransferase I (GnTI, encoded by the Mgat1 gene) controls the synthesis of paucimannose N-glycans. Either deletion or neuron-specific knockdown of Mgat1 in wild type flies results in pronounced defects in locomotion, structural defects in the adult central nervous system and a severely reduced lifespan. We have recently shown that neuronal expression of a wild-type Mgat1 transgene in Mgat1-null flies rescues the structural defects in the brain (fused β-lobes) and the shortened lifespan and, surprisingly, results in a dramatic 135% increase in mean lifespan relative to genetically identical controls that do not express the transgene. In this review, we discuss various approaches that can be used to determine the roles of paucimannose N-glycans in Drosophila longevity and in the adult CNS.     

Role of glycosylation in transport of vesicular stomatitis virus envelope glycoprotein. A new class of mutant defective in glycosylation and transport of G protein

A temperature-sensitive mutant (ts gamma 1) of the Cocal serotype of vesicular stomatitis virus synthesizes at the permissive temperature (32 degrees C) a glycoprotein G whose size is smaller (Mr 68,000) than the wild-type (Mr 71,000) and that renders the virion thermolabile. At the nonpermissive temperature (39 degrees C), reduced amounts of noninfectious virus-like particles deficient in G protein were produced. The size of the intracellular G protein was further decreased (Mr 64,000) at the nonpermissive temperature. Biochemical studies including sugar labeling, tryptic peptide analysis, and NH2-terminal sequence analysis of the various glycoproteins suggest that at 32 degrees C a G protein containing a single glycosidic moiety is synthesized. The G protein containing only 1 oligosaccharide residue is transported to the cell surface and is incorporated in infectious virus particles. In contrast, the G protein synthesized at 39 degrees C is nonglycosylated and fails to reach the cell surface. These results suggest that glycosylation of G protein is essential for its transport to the cell surface, and the presence of a single carbohydrate chain is sufficient for this purpose.     

Expression of the sweet protein brazzein in maize for production of a new commercial sweetener

The availability of foods low in sugar content yet high in flavour is critically important to millions of individuals conscious of carbohydrate intake for diabetic or dietetic purposes. Brazzein is a sweet protein occurring naturally in a tropical plant that is impractical to produce economically on a large scale, thus limiting its availability for food products. We report here the use of a maize expression system for the production of this naturally sweet protein. High expression of brazzein was obtained, with accumulation of up to 4% total soluble protein in maize seed. Purified corn brazzein possessed a sweetness intensity of up to 1200 times that of sucrose on a per weight basis. In addition, application tests demonstrated that brazzein-containing maize germ flour could be used directly in food applications, providing product sweetness. These results demonstrate that high-intensity sweet protein engineered into food products can give sweetener attributes useful in the food industry.     

A new role for ATM

The various pathologies in ataxia telangiectasia (A-T) patients including T-cell lymphomagenesis have been attributed to defects in the DNA damage response pathway because ATM, the gene mutated in this disease, is a key mediator of this process. Analysis of Atm-deficient thymocytes in mice reveals that the absence of this gene results in altered mitochondrial homeostasis, a phenomenon that appears to result from abnormal mitophagy engagement. Interestingly, allelic loss of the autophagic gene Becn1 delays tumorigenesis in Atm-null mice presumably by reversing the mitochondrial abnormalities and not by improving the DNA damage response (DDR) pathway. Thus, ATM plays a critical role in modulating mitochondrial homeostasis perhaps by regulating mitophagy.