原核生物糖基转移酶PglL的研究进展

原核生物蛋白质糖基化修饰系统包括N-糖基化和O-糖基化两种,其中O-糖基化修饰系统中的寡糖基转移酶PglL对底物和糖链的特异性更低,在利用蛋白糖基化制备多糖结合疫苗中应用范围更广。简要综述了PglL的基本结构及其在糖基化反应中的特异性,为以后PglL的应用提供初步指导。     

原核生物糖基化修饰系统及其应用前景

传统认为只有真核生物才有蛋白质糖基化修饰现象,虽然在原核生物细胞中发现糖蛋白的存在已经有数十年,但是没有引起我们足够的重视。最近,在细菌中发现了蛋白质的糖基化修饰系统,最具代表性的是空肠弯曲弧菌的N-糖基化修饰系统、脑膜炎奈瑟球菌和绿脓杆菌的O-糖基化修饰系统。这些糖基化修饰系统已成功地转移到大肠杆菌中,并且独立发挥其糖基化修饰作用。寡糖转移酶在修饰过程中起关键作用,且寡糖转移酶对糖底物的特异性要求非常低,这使得按照我们的需求来"定制糖蛋白"成为可能,并标志着"原核生物糖基工程"的到来,这将为糖结合疫苗的发展提供良好的契机。     

放线菌蛋白O-甘露糖基化的研究进展

蛋白质糖基化作为一种翻译后修饰方式不仅在真核生物中广泛存在,而且在原核生物中尤其是放线菌中也存在。本文针对放线菌中的分枝杆菌属、链霉菌属和棒状杆菌属存在的O-甘露糖基化蛋白的聚糖链结构、糖基化过程以及生物学意义加以论述。     

福氏志贺菌O-抗原磷酸乙醇胺修饰研究进展

福氏志贺菌是发展中国家引起痢疾的主要致病菌。福氏志贺菌O-抗原除噬菌体介导的糖基化和(或)乙酰化外,最近发现一种新的修饰方式磷酸乙醇胺修饰,其机制为由质粒携带的opt基因编码磷酸乙醇胺转移酶在O-抗原的鼠李糖II或(和)鼠李糖III上添加磷酸乙醇胺基团,从而形成血清型Xv、4av和Yv福氏志贺菌,并表达MASF IV-1抗原。人工转化携带opt基因的质粒到无MASF IV-1抗原表达的不同血清型福氏志贺菌中,转化菌株都能表达MASF IV-1抗原。在某些血清型福氏志贺菌中,O-抗原的磷酸乙醇胺修饰与糖基化、乙酰化修饰之间相互作用。现对福氏志贺菌的O-抗原磷酸乙醇胺修饰机制及其与糖基化、乙酰化修饰间的相互作用进行了综述。     

O-糖基化位点预测及糖基化酶催化特点

O-糖链维持所连接蛋白质部分的空间构象。O-糖基化作为生物体内重要的生物过程,其起始步骤具有复杂的高度选择性,迄今为止还未发现固定的模式。人们通过比较已知O-糖基化部位周围的氨基酸序列,推测出O-糖基化位点的一些规律及其酶的催化特性。     

代谢性标记结合二维电泳寻找O-糖基化蛋白的研究

蛋白质的O-糖基化(O-glycosylation)是一种重要的翻译后修饰,参与诸多生理和病理过程。目前,对于蛋白质的O-糖基化的研究进展仍非常缓慢,一个重要的原因就是缺乏高效的对O-糖基化蛋白进行分离和鉴定的技术。创新性地将点击化学反应、二维电泳和质谱技术结合,对寻找细胞内O-糖基化蛋白进行了技术探索性研究。首先利用代谢性标记手段,在人肝癌细胞HCCLM6培养基中加入四乙酰化叠氮半乳糖胺(Ac4GalNAz),对细胞内O-GalNAc糖基化蛋白进行标记;其次通过点击化学将炔基荧光基团连接至标记的O-糖基化蛋白的叠氮基团;应用二维电泳技术对标记蛋白进行分离,并找到13个具有荧光信号的蛋白点;最后对具有荧光信号的蛋白进行质谱鉴定,成功鉴定到7种蛋白,经软件预测后,GRP78蛋白和ANXA1蛋白均具有潜在的O-糖基化位点。这为寻找细胞或生物体中的O-糖基化蛋白奠定了基础,并为高通量筛选O-糖基化蛋白提供技术平台。     

Tn抗原、sTn抗原和T抗原参与肿瘤转移的过程

O-糖基化是蛋白质翻译后修饰的主要方式之一。O-糖基化不仅存在于正常的细胞表面,且肿瘤细胞表面常常伴随异常的O-聚糖。Tn抗原、sTn抗原和T抗原是O-聚糖的重要组成部分,在肿瘤表面过度表达,并参与肿瘤转移的过程。本文就O-聚糖参与肿瘤发生发展的分子机制做一简要综述。     

酵母O-甘露糖转移酶的研究进展

随着糖蛋白类药物的需求量不断增加,酵母表达系统的人源化改造成为当务之急。其中,酵母蛋白的O-糖基化因O-糖链种类繁多、组分单一以及糖基化位点预测困难等因素,限制了酵母蛋白的O-糖基化人源化改造。从酵母蛋白的O-糖链结构、O-糖链发生过程及O-糖链的功能展开综述,重点介绍起始酵母蛋白O-糖基化过程的O-甘露糖转移酶家族(family of protein O-mannosyltransferases, PMTs)成员的研究现状,希望对酵母蛋白O-糖基化人源化改造研究提供参考。     

蛋白分子O-糖基化的研究进展

蛋白分子的氧连接糖基化（O-糖基化）修饰是生物体内必不可少的转录后化学修饰之一,其作用方式类似磷酸化,并且两者之间相互作用,共同调节生物大分子的活性。O-糖基化修饰在生物体的转录、翻译、核运输、细胞骨架的形成以及调节细胞器的功能中发挥着重要的作用。通过影响细胞信号的传导,在细胞吞噬、炎性细胞的迁移以及细胞内大分子物质的循环中也起着重要作用。该文主要通过介绍蛋白分子O-糖基化修饰的基础理论以及。一糖基化修饰作用的几个方面,来简要阐述O-糖基化修饰在生物体内发挥的作用。     

肿瘤细胞O-GalNAc聚糖的生物合成途径

对肿瘤糖生物合成的研究发现肿瘤细胞表面和分泌物中有许多结构和功能变异的O-聚糖糖蛋白。合成O-聚糖的糖基转移酶和磺基转移酶是以相关的酶家族存在的,对这些酶肿瘤调控作用的研究使我们有可能揭示肿瘤形成和糖基化之间的关系;理解和控制肿瘤细胞的反常的生物学行为并利用肿瘤中因异常O-糖基化出现的特定的抗原决定簇,设计出诊断和治疗肿瘤的新方法。     

Database analysis of O-glycosylation sites in proteins

Statistical analysis was carried out to study the sequential aspects of amino acids around the O-glycosylated Ser/Thr. 992 sequences containing O-glycosylated Ser/Thr were selected from the O-GLYCBASE database of O-glycosylated proteins. The frequency of occurrence of amino acid residues around the glycosylated Ser/Thr revealed that there is an increased number of proline residues around the O-glycosylation sites in comparison with the nonglycosylated serine and threonine residues. The deviation parameter calculated as a measure of preferential and nonpreferential occurrence of amino acid residues around the glycosylation site shows that Pro has the maximum preference around the O-glycosylation site. Pro at +3 and/or -1 positions strongly favors glycosylation irrespective of single and multiple glycosylation sites. In addition, serine and threonine are preferred around the multiple glycosylation sites due to the effect of clusters of closely spaced glycosylated Ser/Thr. The preference of amino acids around the sites of mucin-type glycosylation is found likely to be similar to that of the O-glycosylation sites when taken together, but the acidic amino acids are more preferred around Ser/Thr in mucin-type glycosylation when compared totally. Aromatic amino acids hinder O-glycosylation in contrast to N-glycosylation. Cysteine and amino acids with bulky side chains inhibit O-glycosylation. The preference of certain potential sequence motifs of glycosylation has been discussed.     

Basic amino acids as modulators of an O-linked glycosylation signal of the herpes simplex virus type 1 glycoprotein gC: functional roles in viral infectivity

The herpes simplex virus type 1 (HSV-1) glycoprotein gC-1 is engaged both in viral attachment and viral immune evasion mechanisms in the infected host. Besides several N-linked glycans, gC-1 contains numerous O-linked glycans, mainly localized in two pronase-resistant clusters in the N-terminal domain of gC-1. In the present study we construct and characterize one gC-1 mutant virus, in which two basic amino acids (114K and 117R) in a putative O-glycosylation sequon were changed to alanine. We found that this modification did not modify the N-linked glycosylation but increased the content of O-linked glycans considerably. Analysis of the O-glycosylation capacity of wild-type and mutant gC-1 was performed by in vitro glycosylation assays with synthetic peptides derived from the mutant region predicted to present new O-glycosylation sites. Thus the mutant peptide region served as a better substrate for polypeptide GalNAc-transferase 2 than the wild-type peptide, resulting in increased rate and number of O-glycan attachment sites. The predicted increase in O-linked glycosylation resulted in two modifications of the biological properties of mutant virus-that is, an impaired binding to cells expressing chondroitin sulfate but not heparan sulfate on the cell surface and a significantly reduced plaque size in cultured cells. The results suggested that basic amino acids present within O-glycosylation signals may down-regulate the amount of O-linked glycans attached to a protein and that substitution of such amino acid residues may have functional consequences for a viral glycoprotein involving virus attachment to permissive cells as well as viral cell-to-cell spread.     

Structural requirements for additional N-linked carbohydrate on recombinant human erythropoietin

N-Linked glycosylation is a post-translational event whereby carbohydrates are added to secreted proteins at the consensus sequence Asn-Xaa-Ser/Thr, where Xaa is any amino acid except proline. Some consensus sequences in secreted proteins are not glycosylated, indicating that consensus sequences are necessary but not sufficient for glycosylation. In order to understand the structural rules for N-linked glycosylation, we introduced N-linked consensus sequences by site-directed mutagenesis into the polypeptide chain of the recombinant human erythropoietin molecule. Some regions of the polypeptide chain supported N-linked glycosylation more effectively than others. N-Linked glycosylation was inhibited by an adjacent proline suggesting that sequence context of a consensus sequence could affect glycosylation. One N-linked consensus sequence (Asn123-Thr125) introduced into a position close to the existing O-glycosylation site (Ser126) had an additional O-linked carbohydrate chain and not an additional N-linked carbohydrate chain suggesting that structural requirements in this region favored O-glycosylation over N-glycosylation. The presence of a consensus sequence on the protein surface of the folded molecule did not appear to be a prerequisite for oligosaccharide addition. However, it was noted that recombinant human erythropoietin analogs that were hyperglycosylated at sites that were normally buried had altered protein structures. This suggests that carbohydrate addition precedes polypeptide folding.     

Analysis of post-translational modification of mycobacterial proteins using a cassette expression system

A recombinant expression system was developed to analyse sequence determinants involved in O-glycosylation of proteins in mycobacteria. By expressing peptide sequences corresponding to known glycosylation sites within a chimeric lipoprotein construct, amino acids flanking modified threonine residues were found to have an important influence on glycosylation. The expression system was used to screen mycobacterial sequences selected using a neural network (NetOglyc) trained on eukaryotic O-glycoproteins. Evidence of glycosylation was obtained for eight of 11 proteins tested. The results suggest that sites involved in O-glycosylation of mycobacterial and eukaryotic proteins share similar structural features.     

A novel model to predict O-glycosylation sites using a highly unbalanced dataset

In silico approaches have become an alternative method to study O-glycosylation. In this paper, we developed a linear interpretable model for O-glycosylation prediction based on an unbalanced dataset, analyzing the underlying biological knowledge of glycosylation. A training set of 4446 sites involving 468 positive sites and 3978 negative sites was developed during this research. The sites were encoded using the amino acid index (AAindex), and the forward stepwise procedure utilized for feature selection. The linear discriminant analysis with an equal a priori probability (PP-LDA) was employed to develop the interpretable model. Performance of the model was verified using both the internal leave-one-out cross-validation and external validation methods. Two non-linear algorithms, the supervised support vector machine and the unsupervised self-organizing competitive neural network, were used as comparisons. The PP-LDA model exhibited improved classification results with accuracy of 82.1?% for cross-validations and 80.3?% for external prediction. Further analysis of this linear model indicated that the properties at position R(1) and the properties relative to hydrophobicity contributed more to the glycosylation prediction. However, the alpha and turn propensities at the C-terminal, together with physicochemical properties at the N-terminal, are also relative to the glycosylation activity. This model is not only capable of predicting the possibility of glycosylation using an unbalanced dataset, but is also helpful to understand the underlying biological mechanisms of glycosylation. Considering the publicly accessibility of our prediction model, a downloadable program is provided in our supply materials.     

Mucin core O-glycosylation is modulated by neighboring residue glycosylation status. Kinetic modeling of the site-specific glycosylation of the apo-porcine submaxillary mucin tandem repeat by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases T1 and T2

The influence of peptide sequence and environment on the initiation and elongation of mucin O-glycosylation is not well understood. The in vivo glycosylation pattern of the porcine submaxillary gland mucin (PSM) tandem repeat containing 31 O-glycosylation sites (Gerken, T. A., Gilmore, M., and Zhang, J. (2002) J. Biol. Chem. 277, 7736-7751) reveals a weak inverse correlation with hydroxyamino acid density (and by inference the density of glycosylation) with the extent of GalNAc glycosylation and core-1 substitution. We now report the time course of the in vitro glycosylation of the apoPSM tandem repeat by recombinant UDP-GalNAc:polypeptide alpha-GalNAc transferases (ppGalNAc transferase) T1 and T2 that confirm these findings. A wide range of glycosylation rates are found, with several residues showing apparent plateaus in glycosylation. An adjustable kinetic model that reduces the first-order rate constants proportional to neighboring glycosylation status, plus or minus three residues of the site of glycosylation, was found to reasonably reproduce the experimental rate data for both transferases, including apparent plateaus in glycosylation. The unique, transferase-specific, positional weighting constants reveal information on the peptide/glycopeptide recognition site for each transferase. Both transferases displayed high sensitivities to neighboring Ser/Thr glycosylation, whereas ppGalNAc T2 displayed additional high sensitivities to the presence of nonglycosylated Ser/Thr residues. This is the first demonstration of the ability to model mucin O-glycosylation kinetics, confirming that under the appropriate conditions neighboring glycosylation status can be a significant factor modulating the first step of mucin O-glycan biosynthesis.     

Bacterial glycoproteins: functions,biosynthesis and applications

Although widely distributed in eukaryotic cells glycoproteins appear to be rare in prokaryotic organisms. The prevalence of the misconception that bacteria do not glycosylate their proteins has been a subject matter of discussion for a long time. Glycoconjugates that are linked to proteins or peptides, generated by the ribosomal translational mechanism have been reported only in the last two to three decades in a few prokaryotic organisms. Most studied prokaryotic glycoproteins are the S-layer glycoproteins of Archeabacteria. Apart from these, membrane-associated, surface-associated, secreted glycoproteins and exoenzymes glycoproteins are also well documented in both, Archea and Eubacteria. From the recent literature, it is now clear that prokaryotes are capable of glycosylating proteins. In general, prokaryotes are deprived of the cellular organelles required for glycosylation. In prokaryotes many different glycoprotein structures have been observed that display much more variation than that observed in eukaryotes. Besides following similar mechanisms in the process of glycosylation, prokaryotes have also been shown to use mechanisms that are different from those found in eukaryotes. The knowledge pertaining to the functional aspects of prokaryotic glycoproteins is rather scarce. This review summarizes developments and understanding relating to characteristics, synthesis, and functions of prokaryotic glycoproteins. An extensive summary of glycosylation that has been reported to occur in bacteria has also been tabulated. Various possible applications of these diverse biomolecules in biotechnology, vaccine development, pharmaceutics and diagnostics are also touched upon.     

蛋白质药物糖基化工程化改造研究进展

多种哺乳和非哺乳动物的蛋白质表达系统已成功用于重组糖蛋白药物的生产。糖基化对于生物药品的研究开发至关重要,对生物药品的药效、半衰期及抗原性等产生重要影响。糖基化工程的目的是生产组分明晰、结构均一的N-和O-连接的糖基化蛋白药物。N-糖基化改造的相关研究显示,利用哺乳动物和非哺乳动物表达系统可以表达均匀的N-聚糖重组糖蛋白。与N-糖基化改造相比, O-糖基化的改造研究尚处于起步阶段。首个糖基化工程单克隆抗体已在美国和日本获得上市批准。综述了重组蛋白表达系统的糖基化工程化改造的研究进展,包括蛋白质药物的 N-糖基化改造和O-糖基化改造的最新进展,以期为蛋白质药物的糖基化工程改造研究提供参考。     

The AATPAP sequence is a very efficient signal for O-glycosylation in CHO cells

The peptide signal sequence for protein O-glycosylation is not fully characterized, although a recent in vitro study proposed that the sequence motif, XTPXP, serves as a signal for mucin-type O-glycosylation. Here, we show that the AATPAP sequence acts as an efficient O-glycosylation signal, in vivo. A secreted fibroblast growth factor (secFGF) was used as a model to analyze glycosylation and its effects on the biological activity of FGF. Two constructs encoding [AATPAP]secFGF in which AATPAP was introduced at the N- or C-terminus of secFGF were constructed in a eukaryotic expression vector. [AATPAP]secFGF proteins were then expressed in Chinese hamster ovary (CHO) cells and secreted into the surrounding medium, primarily as modified forms sensitive to sialidase but not to peptide N-glycosidase F. The modifying groups were not seen when the AATPAP sequence was converted to AAAPAP or when [AATPAP]secFGF was expressed in mutant cells incapable of UDP-GalNAc biosynthesis. The results indicate that the modifying groups were mucin-type O-glycans and that the AATPAP served as an efficient O-glycosylation signal sequence. The O-glycosylated forms of [AATPAP]secFGF were as mitogenic toward human vascular endothelial cells as unmodified secFGF, suggesting that introduction of the signal into biologically active polypeptides is a promising approach with which O-glycosylation may be achieved without affecting original activity.     

Early steps in O-linked glycosylation and clustered O-linked glycans of herpes simplex virus type 1 glycoprotein C: effects on glycoprotein properties

The pathogenesis of herpes simplex virus type 1 (HSV-1) implies the sequential infection of many cell types from mucosal cells to neurons, each having a unique pattern of protein glycosylation. The HSV-1 glycoprotein gC-1 is highly glycosylated and contains not only N-linked glycans but also a large number of O-linked glycans, some of which are clustered into two pronase-resistant arrays in the vicinity of the HSV-1 receptor-binding domain of gC-1. The aim of the present study was to characterize gC-1 signals for addition of clustered glycans, to determine the efficacy of synthetic peptides, representing putative O-glycosylation signals, as substrates for a panel of GalNAc transferases, and to identify possible effects of early O-linked glycosylation on the biological functions of gC-1. Gel filtration analysis of the pronase-resistant gC-1 O-glycan clusters from a glycoprotein mutant, lacking a site for N-linked glycosylation at Asn 73 in the vicinity of the O-glycosylation signal, suggested that one function of this N-linked glycan was to modulate the access for GalNAc transferases to one particular O-glycosylation peptide signal (aa 80-104). The ability of four GalNAc-transferase isoenzymes with different cell type expression patterns to initialize O-glycosylation of synthetic gC-1 derived peptides was analyzed. Two synthetic gC-1 peptides (aa 55-69 and aa 80-104) were excellent substrates for all four GalNAc-transferases, suggesting that cell types expressing less frequent GalNAc transferase species with unusual acceptor peptide sequence specificities may also produce a highly O-glycosylated gC-1 after HSV-1 infection. The O-linked glycans were not essential for cell surface expression of gC-1, but monoclonal antibody-assisted epitope analysis of N-acetylgalactosaminidase-treated gC-1 showed that the O-linked monosaccharide GalNAc contributed to expression of a three-dimensional epitope overlapping the heparan sulfate-binding domain of gC-1.