A serial lectin approach to the mucin-type O-glycoproteome of Drosophila melanogaster S2 cells

Identification of mucin-type O-glycosylated proteins with known functions in model organisms like Drosophila could provide keys to elucidate functions of the O-glycan moiety and proteomic analyses of O-glycoproteins in higher eukaryotes remain a challenge due to structural heterogeneity and a lack of efficient tools for their specific isolation. Here we report a strategy to evaluate the O-glycosylation potential of the embryonal hemocyte-like Drosophila Schneider 2 (S2) cell line by expression of recombinant glycosylation probes derived from tandem repeats of the human mucin MUC1 or of the Drosophila salivary gland protein Sgs1. We obtained evidence that mucin-type O-glycosylation in S2 cells grown under serum-free conditions is restricted to the Tn-antigen (GalNAcalpha-Ser/Thr) and the T-antigen (Galbeta1-3GalNAcalpha-Ser/Thr) and this structural homogeneity enables unique glycoproteomic strategies. We present a label-free strategy for the isolation, profiling and analysis of O-glycosylated proteins consisting of serial lectin affinity capture, 2-DE-based glycoprotein analysis by O-glycan specific mAbs and protein identification by MALDI-MS. Protein identity and O-glycosylation was confirmed by ESI-MS/MS with detection of diagnostic sugar oxonium-ion fragments. Using this strategy, we established 2-D reference maps and identified 21 secreted and intracellular mucin-type O-glycoproteins. Our results show that Drosophila S2 cells express O-glycoproteins involved in a wide range of biological functions including proteins of the extracellular matrix (Laminin gamma-chain, Peroxidasin and Glutactin), pathogen recognition proteins (Gnbp1), stress response proteins (Glycoprotein 93), secreted proteases (Matrix-metalloprotease 1 and various trypsin-like serine proteases), protease inhibitors (Serpin 27 A) and proteins of unknown function.     

Synthesis of fagopyritols A1 and B1 from D-chiro-inositol

Fagopyritol A1 (3-O-alpha-d-galactopyranosyl-d-chiro-inositol) and fagopyritol B1 (2-O-alpha-d-galactopyranosyl-d-chiro-inositol) have been synthesized by glycosylation of the diequatorial diol 1,4,5,6-tetra-O-benzoyl-d-chiro-inositol, readily obtained from d-chiro-inositol, with 2,3,4,6-tetra-O-benzyl-d-galactopyranosyl trichloroacetimidate.     

A novel CFTR disease-associated mutation causes addition of an extra N-linked oligosaccharide

We have examined the influence of a novel missense mutation in the fourth extracytoplasmic loop (EL4) of CFTR detected in a patient with cystic fibrosis. This substitution (T908N) creates a consensus sequence (N X S/T) for addition of an N-linked oligosaccharide chain near the C-terminal end of EL4. Oligosaccharyl transferase generally does not have access to this consensus sequence if it is closer than about twelve amino acids from the membrane. However, the T908N site is used, even though it is within four residues of the predicted membrane interface and the oligosaccharide chain added binds calnexin, a resident chaperone of the ER membrane. The chloride channel activity of this variant CFTR is abnormal as evidenced by a reduced rate of ³⁶Cl^– efflux and a noisy single channel open state. This may reflect some displacement of the membrane spanning sequence C-terminal of EL4 since it contains residues influencing the ion pore.     

Glycan Antagonists and Inhibitors: A Fount for Drug Discovery

ABSTRACT

Glycans, the carbohydrate chains of glycoproteins, proteoglycans, and glycolipids, represent a relatively unexploited area for drug development compared with other macromolecules. This review describes the major classes of glycans synthesized by animal cells, their mode of assembly, and available inhibitors for blocking their biosynthesis and function. Many of these agents have proven useful for studying the biological activities of glycans in isolated cells, during embryological development, and in physiology. Some are being used to develop drugs for treating metabolic disorders, cancer, and infection, suggesting that glycans are excellent targets for future drug development.     

The Role of N53Q Mutation on the Rat μ-Opioid Receptor Function

Glycosylation of the μ-opioid receptor may play an important role on its function. Using nested PCR, N53Q mutation was prepared in the N-terminal region of the rat μ-opioid receptor cDNA and cloned into the pcDNA3 vector. The plasmids containing the wild-type and mutated receptor cDNA were transfected into the COS-7 cells. Intracellular cAMP was measured in the morphine-treated and untreated transfected cells using an ELISA kit. Plasmid expression was evaluated using X-gal staining. Intracellular concentration of cAMP in the N53Q-mutated cells was not significantly different from the wild-type. The expression of the transfected plasmids was confirmed. Therefore, based on these results, it seems that glycosylation at the N53 site of the rat μ-opioid receptor does not influence the function of this receptor significantly.     

Enzymatic conversion of proteins to glycoproteins by lipid-linked saccharides: A study of potential exogenous acceptor proteins

Previous studies have shown that a membrane preparation from hen oviduct catalyzes transfer of oligosaccharide from oligosaccharide-P-P-dolichol to denatured RNase and α-lactalbumin. To gain further insight into the structural requirements of a protein that allow it to serve as a substrate for glycosylation, the acceptor ability of a variety of other modified proteins containing the tripeptide sequence -ASN-X-(SER/THR)- has been investigated. Of 7 proteins tested, 2 (ovine prolactin and rabbit muscle triosephosphate isomerase) could be enzymatically glycosylated by a particulate preparation from hen oviduct. The remaining 5 proteins, assayed as either S-carboxy-methylated or S-aminoethylated derivatives, were inactive as carbohydrate acceptors. However, cyanogen bromide treatment of 2 of the inactive proteins, bovine catalase and concanavalin A from jack bean, yielded peptide fragments which served as substrates for glycosylation. These results suggests that for some proteins, disruption of the tertiary structure is sufficient to allow attachment of carbohydrate. Other denatured proteins may possess additional restrictions imposed by their secondary structure. In certain cases, these restrictions are removed when the polypeptide chain is fragmented.     

Glycoprotein synthesis in lysolecithin-treated cells using sugar nucleotides as glycosyl donors

Summary The 3T3 cells were treated with 50 μg/ml lysolecithin (LL) followed by the addition of exogenously supplied radiolabelled sugar nucleotides to serve as direct glycosyl donors. These were found to be 1.5 to 3.0 times more active than untreated cells in their glycosyl transferase activities depending on the particular sugar nucleotide used. Mannosyl transferase activity was not inhibited by 2-deoxyglucose or mannose-1-phosphate, indicating that the sugar nucleotide remained intact throughouth the assay period. Preincubation of the cells with tunicamycin caused an 85% decrease in mannosyl transfer, which suggested that the normal pathway of glycosylation via lipid intermediates was still operable in the treated cells. Fractionation of control and LL-treated cells after incubation with UDP[³H]galactose revealed that only microsomal and cytosolic proteins from the treated cells were radioactive. Thus, intracellular labelling of permeabilized cells was allowed. About 80% of the radiolabeled product was glycoprotein in nature, based upon its solubilization with pronase. This work was supported by institutional funds granted by Texas Woman's University.     

A plant signal sequence enhances the secretion of bacterial ChiA in transgenic tobacco

When the secreted bacterial protein ChiA is expressed in transgenic tobacco, a fraction of the protein is glycosylated and secreted from the plant cells; however most of the protein remains inside the cells. We tested whether the efficiency of secretion could be improved by replacing the bacterial signal sequence with a plant signal sequence. We found the signal sequence and the first two amino acids of the PR1b protein attached to the ChiA mature protein directs complete glycosylation and secretion of the ChiA from plant cells. Glycosylation of this protein is not required for its efficient secretion from plant cells.     

Glycosylated Haemoglobin (Hb A1) in Normal Rhesus Monkeys (Macaca mulatta)

Glycosylated haemoglobins were measured in 23 healthy juvenile rhesus monkeys by the use of commercially available minicolumn chromatography (Quick Sep., Isolab Inc., Ohio, USA) to establish the normal range. Values obtained (mean ± 1 standard deviation (SD) 1.57 ± 0.68%) were significantly lower than that of 17 adult healthy human volunteers by the use of the same method of estimation (mean ± 1 SD of 5.34 ± 0.78%).     

Cloning and characterization of a candidate nutritive glycoprotein from the albumen gland of the freshwater snail, Helisoma duryi (Mollusca: Pulmonata)

Abstract. The albumen gland is a female accessory sex gland that synthesizes and secretes perivitelline fluid around pulmonate eggs. The perivitelline fluid is composed of mainly galactogen and proteins, and is thought to provide nourishment to the embryos during development. We have previously identified the major secretory protein of the albumen gland of the freshwater snail Helisoma duryi as a native glycoprotein of ∼288 kDa, consisting of four 66-kDa subunits. In this study, the major albumen gland protein in H. duryi was purified, cloned, and the full-length cDNA sequence determined. Nucleotide sequence analysis revealed that the albumen gland protein (HdAGP) shared 83% identity with a partial cDNA sequence from a developmentally regulated albumen gland protein in Biomphalaria glabrata . The HdAGP mRNA was detected by RT-PCR in the albumen gland, ovotestis, mantle and digestive gland. SDS-PAGE analysis of the albumen gland protein in egg masses at different stages of development showed that the amount of HdAGP steadily decreased during embryogenesis, suggesting its possible catabolism by the developing embryos. Protein domain searches suggested that the HdAGP shared limited sequence identity, and adopted a similar three-dimensional conformation to the bactericidal, permeability increasing, protein family, raising the possibility of a potential bactericidal function for this important reproductive/developmental protein.     

A one pot synthesis of mono- and di-lactosyl sphingosines

The importance of analogues of lactosyl ceramides as basic structures of many natural glycosphingolipids provided a rationale for developing an efficient synthetic route to these compounds. We report herein a novel approach to synthesize several members of this family. Glycosylation of N-diphenylmethylene-spingosine, which exists in an imine–oxazolidine tautomeric mixture, with acetobromolactose under a modified Koenigs-Knorr condition yielded lactosyl -(1 1) sphingosine, lactosyl -(1 3) sphingosine and dilactosyl sphingosine in good yields. A similar glycosylation could be applicable to the synthesis of other glycosphingolipids.     

水稻N-糖酰胺酶基因 (OsPNGase A) 的克隆及在毕赤酵母中的分泌表达

N-聚糖酶是一类广泛应用于糖蛋白的N-糖基化修饰研究中的去糖基化酶。本研究通过RT-PCR从水稻中克隆了一个高GC含量(69.48%)的N-聚糖酶基因(Os PNGase A,XM_015775832),通过无缝克隆技术构建酵母分泌型表达载体p PICZ(α)A-Os PNGase A,在毕赤酵母SMD1168H中进行诱导表达,发酵液经DEAE Sepharose阴离子交换层析和His Trap HP金属离子螯合层析纯化,产量可达到12.3 mg/L,比活力为258 U/mg。SDS-PAGE结果显示,纯化的Os PNGase A为单一条带且与预期分子量一致。Os PNGase A能作用于水稻中重组表达的人转铁蛋白(TRF)、玉米中重组表达的鸡蛋抗生物素蛋白(Avidin)以及辣根过氧化物酶(HRP),并且对Avidin的酶切效果优于商业化的PNGase F。Os PNGase A反应的最适p H和温度分别为p H 6.0和40℃,在中性和碱性以及含有100 mmol/L还原剂β-ME和DTT的条件下仍具有活性。水稻Os PNGase A的成功表达为植物糖蛋白的研究提供一个新的工具酶,酵母分泌表达体系的建立为PNGase A的大量制备奠定了基础。     

A functional antibody lacking N-linked glycans is efficiently folded, assembled and secreted by tobacco mesophyll protoplasts

A potential drawback in the use of plants as an expression platform for pharmaceutical proteins such as antibodies is that plant-specific N-glycosylation can result in proteins with altered function and potential antigenicity. In many cases, the N-glycans are essential for the correct folding, assembly and transport of the recombinant proteins. We tested whether progressive removal of glycosylation sites had a detrimental effect on the synthesis, assembly and secretion of a plant-made immunoglobulin G, Guy's 13. Our results indicate that the plant secretory pathway can cope well with aglycosylated antibody chains. The immunoglobulin without N-linked glycans is correctly assembled and secreted by tobacco protoplasts. Capture enzyme-linked immunosorbent assay also shows that antigen-binding properties are unaffected. Our results therefore suggest one possible alternative to the engineering of a humanized glycosylation machinery in plants.     

糖基转移酶和去糖基化酶

在糖基化工程中,通过酶法对蛋白质进行糖基化修饰和对天然糖蛋白去糖基化是研究糖蛋白结构与功能的重要手段。本文综述了近年来所纯化的主要的糖基化转移酶和去糖基化酶的性质和应用。     

A modified storage protein is synthesized,processed, and degraded in the seeds of transgenic plants

In vitro mutagenesis was used to supplement the sulfur amino acid codon content of a gene encoding -phaseolin, a Phaseolus vulgaris storage protein. The number of methionine codons in the phaseolin gene was increased from three to nine by insertion of a 45 base pair (bp) synthetic duplex. Either modified or normal phaseolin genes were integrated into the genome of tobacco plants through Agrobacterium tumefaciens-mediated transformation. Although similar levels of phaseolin RNA are detected in seeds of plants transformed with either the normal or modified (himet) gene, the quantity of himet protein is consistently much lower than normal -phaseolin. Himet phaseolin is expressed in a temporal- and organ-specific fashion, and is N-glycosylated and assembled into trimers in the manner of normal phaseolin. After germination, both types of phaseolin are hydrolyzed, but the himet protein is more quickly degraded. Electron microscopic immunocytochemical observations of developing seeds indicate that the himet protein is primarily localized in the endoplasmic reticulum (ER) and in Golgi apparatus secretion vesicles. Himet phaseolin is absent from protein storage vacuoles, termed protein bodies, where normal phaseolin is deposited in transgenic tobacco. We interpret the immunocytochemical data to indicate that himet phasolin is transported through the ER and Golgi apparatus and is then degraded in Golgi secretion vesicles or the protein bodies.Mention of trademark, proprietary product, or vendor does not constitute a guarantee of warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable. Publication No. 70 from Agrigenetics Advanced Science Company.     

糖基因Glt8d2重组蛋白的表达、纯化及多克隆抗体的制备

目的构建新糖基转移酶基因Glt8d2的原核表达载体,诱导融合蛋白的表达,并对其进行纯化;制备兔抗GltSd2蛋白多克隆抗体并进行鉴定。方法应用RT—PCR技术,以HepG2细胞mRNA为模板,扩增Glt8d2目的基因片段,构建原核表达载体pET-32a（＋）-Glt8d2。转化大肠埃希菌B121,异丙基-D-半乳糖苷诱导并通过SDS—PAGE凝胶电泳分析、Western印迹分析、生物质谱分析证实Gh8d2重组蛋白表达正确。大量表达后利用Ni’亲和柱对表达蛋白进行纯化及柱上复性。纯化蛋白免疫新西兰大白兔,获得抗Gh8d2蛋白的多克隆抗体。以纯化的Glt8d2重组蛋白为抗原,分别以免疫前后的新西兰兔血清作为第一抗体,利用Western印迹和酶联免疫吸附法对多克隆抗体进行特异性分析及效价检测。结果扩增获得Glt8d2基因片段,成功表达了Gh8d2重组蛋白,经SDS—PAGE凝胶电泳和Western印迹分析得到证实。成功获得重组蛋白及兔抗Gh8d2多克隆抗体。ELISA检测证实多克隆抗体效价〉1：320000。免疫组织化学分析显示,该基因编码的糖基转移酶在肝实质细胞内呈胞质模式表达分布。结论利用大肠埃希菌B121能够成功表达Gh8d2重组蛋白,获得高特异性、高效价兔抗Glt8d2重组蛋白的多克隆抗体,为今后研究Glt8d2基因的生物学功能研究奠定了基础。     

Differential glycosylation of N-POMC1-77 regulates the production of gamma 3-MSH by purified pro-opiomelanocortin converting enzyme. A possible mechanism for tissue-specific processing.

The amino terminus of bovine pro-opiomelanocortin (N-POMC^1–77) is partially processed in the intermediate lobe of the pituitary to N-POMC^1–49 and lys-γ₃ -melanotropin. Two pools of N-POMC^1–77 were isolated which were differentially glycosylated at threonine⁴⁵, while N-POMC^1–49 isolated from bovine intermediate lobe extracts existed in a non-glycosylated form. This suggested that differential O-linked glycosylation of N-POMC^1–77 may regulate cleavage at the Arg⁴⁹-Lys⁵⁰ processing site. We tested this hypothesis by incubating N-POMC^1–77 glycoforms with purified pro-opiomelanocortin converting enzyme. Only non-O-glycosylated N-POMC^1–77 and O-glycosylated N-POMC^1–77 with truncated oligosaccharide sidechains were sensitive to cleavage and generated predominantly lys-γ₃ -melanotropin, identified by high-performance liquid chromatography. These data provide the first functional evidence to support a role for differential O-linked glycosylation in the regulation of the processing of the N-terminus of bovine POMC.