汉坦病毒包膜糖蛋白的研究进展

汉坦病毒(Hantavirus,HV)是人类重要的致病病毒,主要引起肾综合征出血热和汉坦病毒肺综合征.HV基因组为分节段的单股负链RNA,由长(L)、中(M)、短(S)三个基因片段组成,分别编码L蛋白、包膜糖蛋白Gn、Gc和核衣壳蛋白N.其生物学活性主要来自Gn、Gc,本文系统阐述了包膜糖蛋白的基因结构和功能,对细胞融合与糖基化这两方面进行了重点叙述.     

副粘病毒融合蛋白分子上特异性细胞融合作用位点的初步确定

目的　找到副粘病毒融合蛋白 (F)分子上与血凝素 神经氨酸酶 (HN)相互作用的特异性区域 ,弄清F融合细胞的分子机理。方法　采用基因定点突变法 ,创造一个酶切位点 ,得到突变株 ,然后用基因重组的方法得到各种重组株 ,并于真核细胞内进行表达 ,Gimsa染色和指示基因法检测细胞融合功能 ,荧光强度分析 (FACS)检测表达效率情况。结果　在将各有关F基因片段进行交换之前 ,创造一个酶切位点时所得突变株的细胞融合功能与野毒株相同 ;将新城疫病毒 (NDV)F和副流感病毒 (PIV)F在膜穿入部分和躯干部分交接处交换时 ,不影响细胞融合功能 ;进一步将F2进行交换时 ,也不影响细胞融合功能 ;而将F的头部进行交换时 ,则检测不到细胞融合作用 ,FACS分析表明 ,F没有达到细胞表面。结论　F分子上与HN相互作用的特异性区域位于膜穿入部分和躯干部分交接处与F1和F2交接处之间 ,即F1除去膜穿入部分和尾部的剩余部分。     

CD45RO N-糖基化位点对galectin-3结合CD45RO突变体转染细胞的影响

目的 构建CD45RO不同N-糖基化位点突变的T细胞株,并检测其与galectin-3的结合情况.方法 采用N→Q定点突变技术分别去除CD45RO的11个N-糖基化位点,制备单个N-糖基化位点缺失的CD45RO,然后将其导入慢病毒表达载体pWPXL中,11个携带单个N-糖基化位点缺失的CD45RO重组慢病毒质粒与慢病毒包装质粒psPAX2和MD2.G共转染293T细胞,将包装重组慢病毒感染CD45-的J45.01细胞,流式分选后获得11株分别表达CD45RO单个N-糖基化位点缺失的J45.01 T细胞株.通过RT-PCR和流式细胞术分别从RNA水平和蛋白水平验证CD45RO突变体的转录和表达,应用流式细胞术检测CD45RO突变细胞株与galectin-3的结合情况.结果 成功构建了1 1个CD45RO单个N-糖基化位点缺失的T细胞株,各突变细胞株能稳定表达突变基因,经测序再次证实突变位点正确,CD45 RO突变体能稳定表达于细胞表面.galectin-3与N327Q突变细胞的结合明显增强,与N36Q突变细胞和N217Q突变细胞的结合明显减弱.结论 CD45RON-糖基化位点对galectin-3与CD45 RO-J45.01细胞的结合有调节作用.     

IgG N-糖基化修饰在糖尿病视网膜病变中作用的研究进展

糖尿病视网膜病变(DR)是免疫炎性疾病,免疫球蛋白G(IgG) N-糖基化修饰与DR密切相关。其中,IgG的半乳糖基化修饰、岩藻糖基化修饰、唾液酸化和平分型N-乙酰葡糖胺修饰发生变化,导致机体处于促炎状态,诱导视网膜发生病变。     

球状脂联素上调脂联素受体1抑制晚期糖基化终产物诱导人脐静脉内皮细胞凋亡的初步研究

目的:观察球状脂联素(gAd)对晚期糖基化终产物(AGEs)诱导人脐静脉内皮细胞(HUVECs)凋亡时脂联素受体1(AdipoR1)表达的影响。方法:体外培养HUVECs,用不同浓度AGEs作用HUVECs,以及预处理gAd后再联合AGEs作用HUVECs。MTT比色法测定细胞活性、Annexin V-FITC/PI双染标记流式细胞仪检测细胞凋亡、实时定量RT-PCR检测内皮细胞AdipoR1 mRNA的表达。结果:AGEs作用HUVECs,细胞凋亡具有AGEs浓度依赖性(P0.05)。相同浓度AGEs作用HUVECs,有和无gAd预处理的2组比较,用gAd预处理组明显拮抗HUVECs凋亡(P0.01)。实时定量RT-PCR检测发现,gAd具有上调AdipoR1 mRNA表达的作用,与相同浓度AGEs作用的无gAd预处理组比较,差异显著(P0.01)。结论:gAd可能通过上调AdipoR1表达拮抗AGEs诱导HUVECs的凋亡。     

CD147在动脉粥样硬化中作用的研究进展

<正>细胞外基质金属蛋白酶诱导剂(Extracellular Matrix Metalloproteinase Inducer,EMMPRIN)-CD147分子是一种高度糖基化的跨膜糖蛋白,属免疫球蛋白超家族成员     

N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸对P38分裂原活化蛋白酶通路调节在肺成纤维细胞胶原合成中的作用

目的:探讨N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸(AcSDKP)是否通过阻断转化生长因子-β1（TGF-β1）介导的P38分裂原活化蛋白酶(MAPK)途径,进而抑制大鼠肺成纤维细胞增殖与Ⅰ型、Ⅲ型胶原蛋白的表达.方法:培养新生大鼠肺成纤维细胞,分为对照组、TGF-β1刺激组、TGF-β受体阻滞剂组、P38MAPK特异性抑制剂组、AcSDKP干预组.采用MTT法检测细胞增殖,免疫细胞化学法检测磷酸化P38蛋白的定位及表达,免疫印迹法检测TGF-β受体、磷酸化P38MAPK、P38MAPK、c-myc及Ⅰ型、Ⅲ型胶原蛋白的表达.结果:与对照组比较,TGF-β1能够促进细胞增殖,而分别给予LY364947、SB203580和AcSDKP干预后,细胞增殖均受到抑制.与对照组比较,TGF-β1刺激组的TGF-β1受体、磷酸化P38MAPK、c-myc以及Ⅰ型、Ⅲ型胶原蛋白表达上调,当分别给予LY364947、SB203580和AcSDKP干预后,肺成纤维细胞TGF-β1受体、磷酸化P38 MAPK、c-myc以及Ⅰ型、Ⅲ型胶原蛋白表达均降低.而各组间比较P38MAPK蛋白表达无明显改变.结论:AcSDKP能够通过阻断TGF-β1介导的P38MAPK信号转导途径,进而抑制肺成纤维细胞增殖和Ⅰ、Ⅲ型胶原蛋白的表达.     

血清内脂素、脂联素和C-反应蛋白在2型糖尿病肾病发生发展中作用的研究

目的 研究血清内脂素、脂联素和C反应蛋白(CRP)在2型糖尿病肾病发生发展中的作用.方法 随机选取2010年3月至2015年12月在我院住院治疗的2型糖尿病肾病患者370例,作为实验组,实验组中根据患者尿蛋白的排泄量分为大量蛋白尿组,微量蛋白尿组,正常蛋白尿组,另外选择100名健康人作为对照组,所有入选者都要进行血糖、血脂的检测.470例入选者体内血清内脂素、脂联素的检测均采用酶联免疫吸附法,C-反应蛋白的检测采用比浊法.结果 实验组中大量尿蛋白组与微量尿蛋白组患者体内血清脂联素的水平与对照组相比明显升高(P＜0.01),而实验组中正常蛋白尿的患者与对照组相比体内血清脂联素水平明显降低.实验组中3组患者体内的血清内脂素与C-反应蛋白水平都明显比对照组升高(P＜0.01).结论 血清内脂素、脂联素和C-反应蛋白参与2型糖尿病肾病的发生、发展,可将患者体内血清内脂素、脂联素和C-反应蛋白的水平作为2型糖尿病肾病评估预后的重要指标.     

N-乙酰基-丝氨酰-天冬氨酰-赖氨酰-脯氨酸对基质金属蛋白酶-1和基质金属蛋白酶组织抑制因子-1的调节在大鼠矽肺纤维化形成中的作用

目的:探讨N-乙酰基-丝氨酰-天冬氨酰-赖氨酰-脯氨酸(AcSDKP)对大鼠肺内基质金属蛋白酶-1(MMP-1)和基质金属蛋白酶组织抑制因子-1(TIMP-1)表达的调节在拮抗矽肺纤维化形成过程中的作用.方法:气管内灌注染尘法制作大鼠矽肺模型,将含有AcSDKP的微量药物释放泵埋入腹腔.实验动物随机分为矽肺模型对照4周组,矽肺模型对照8周组,矽肺模型4周组,矽肺模型8周组,抗纤维化治疗组及预防治疗组.H-E染色和免疫组织化学显色对矽肺纤维化病变和MMP-1和TIMP-1在肺组织内的表达进行形态学观察;免疫印迹法对肺内MMP-1和TIMP-1酶蛋白表达进行检测.结果:与模型对照组比较,矽肺大鼠肺内MMP-1和TIMP-1表达增强.与矽肺模型组比较,AcSDKP能够上调矽肺大鼠肺内MMP-1的表达,下调TIMP-1的表达,使MMP-1/TIMP-1的比值升高.结论:AcSDKP能够促进矽肺大鼠肺内MMP-1的表达,抑制TIMP-1的表达,从而加速了细胞外基质(包括胶原)的降解,这可能与AcSDKP抗矽肺纤维化的作用有关.     

CD54和LFA-1的相互作用在6A8α-甘露糖苷酶表达抑制的JurkatT细胞的黏附性增强中的作用

目的研究CD54和LFA-1的相互作用在6A8 α-甘露糖苷酶表达抑制的Jurkat细胞(AS)的黏附性增强中的作用。方法用细胞凝集试验确证AS细胞间黏附的增强，用细胞与细胞间黏附分子-1-人IgG的Fc片段(ICAM-1-Fc)的黏附试验和阻断性抗CD11a抗体的阻断试验研究CD54-LFA-1的作用，用单克隆抗体MEM-148检测As细胞LFA-1亲和力的变化，用单克隆抗体NKI-L16检测AS细胞LFA-1亲合力的变化，用鬼笔环肽染色细胞骨架，用Jurkat-Raji细胞间的作用作模型研究6A8α-甘露糖苷酶表达抑制对T和B细胞间黏附的影响，用ConA结合试验检测细胞中蛋白质N-糖基化的变化。结果(1)AS细胞间的黏附性增强主要与CD54及CD11a表达的增强相关，也与LFA-1亲和力的增高相关；(2)AS细胞的细胞骨架发生重排；(3)As细胞与Raji细胞间的黏附也增强；(4)ConA与AS细胞的结合增强。结论CD54和LFA-1的相互作用在AS Jurkat T细胞的黏附性增强中起重要作用。细胞骨架重排也可能起作用。As细胞的蛋白质发生了N-糖基化修饰。     

风疹病毒包膜糖蛋白糖基化作用对细胞融合的影响

目的 探讨JR23株风疹病毒包膜糖蛋白E2和E1的糖基化作用对其引起的特异性细胞融合的影响.方法 采用基因定点突变的方法构建6个突变株,分别改变E2或E1蛋白的糖基化位点结构.采用Giemsa染色法与指示基因法检测各突变株蛋白引起的细胞融合,流式细胞术检测其在细胞表面的表达效率,并用血细胞吸附试验检测其吸附活性.结果 与野毒株蛋白相比较,突变株蛋白E2 N53G、S73I、S131V与E1 T78A、T179A、1211A引起的细胞融合效率分别为62.73%、66.66%、55.12%、66.93%、87.33%、90.18%;除了E2 S131V之外,其他突变株蛋白在细胞表面的表达效率均有不同程度的降低;只有E2 S73I与E1 T78A的血细胞吸附效率略有降低.结论 E2蛋白的3个糖基化位点与E1蛋白的N76位糖基化位点的糖基化对细胞融合有重要作用,E1的N177与N209位点糖基化对细胞融合的影响较小.     

肾综合征出血热病毒结构蛋白在患者尿中融合细胞上的表达及其意义

为了探讨汉坦病毒（ＨＶ）结构蛋白与患者尿融合细胞的关系，采用抗ＨＶ包膜结构蛋白Ｇ２的单克隆抗体ＭｃＡｂ－ＬＶ４８Ａ，抗ＨＶ血凝素的ＭｃＡｂ－３Ｄ８，抗ＨＶ核衣壳蛋白的ＭｃＡｂ－Ａ３５等，分别对３０例肾综合征出血热患者尿液中的融合细胞进行免疫酶染色。结果ＭｃＡｂ－ＬＶ４８Ａ阳性率为７０％（２１／３０）；ＭｃＡｂ－３Ｄ８阳性率为７３％（２２／３０）；ＭｃＡｂ－Ａ３５阳性率为７％（２／３０）。该结果提示，患者尿液中的融合细胞的形成与ＨＶ膜结构蛋白在泌尿系统上皮细胞上的表达有密切关系。     

Determinant for the inhibition of ecotropic murine leukemia virus infection by N-linked glycosylation of the rat receptor

Ecotropic murine leukemia viruses (MLVs) recognize the third extracellular loop of the receptor, cationic amino acid transporter type 1 (CAT1). The CAT1 protein contains two conserved N-linked glycosylation sites in the third extracellular loops of the mouse, rat, and hamster receptors (mCAT1, rCAT1, and hCAT1, respectively). Glycosylation of the rCAT1 and hCAT1 receptors inhibits ecotropic MLV infection of CAT1-expressing cells, but that of the mCAT1 does not afford the cells this protection. As compared to the mCAT1 protein, the rCAT1 and hCAT1 proteins possess three and six amino acid insertions, respectively, in the third extracellular loop. To determine whether these inserted amino acids are associated with ecotropic MLV infection inhibition by glycosylation, several mutants of mCAT1 and rCAT1 receptors were constructed. Of all the mutants generated in the present study, only rCAT1 mutant 1 exhibited detectable protein expression levels. The rCAT1 mutant 1-expressing human NP2 cells were more susceptible to transduction by ecotropic MLV vectors than the wild-type rCAT1-expressing cells. Tunicamycin, an N-glycosylation inhibitor, increased transduction titer in the wild-type rCAT1-expressing cells, but did not do so in the cells expressing either the mCAT1 or rCAT1 mutation 1. An amino acid substitution in the glycosylation site of the wild-type rCAT1 conferred higher infection susceptibility, but that of the rCAT1 mutant 1 did not. As with the wild-type mCAT1 and rCAT1 proteins, the rCAT1 mutants were detected on the cell surface by immunofluorescence microscopy. Tunicamycin treatment did not affect cellular distribution of the rCAT1 mutant 1, wild-type mCAT1 or rCAT1 proteins. These results indicate that the extra amino acids in the rCAT1 (as compared to the mCAT1) are associated with inhibition of ecotropic MLV infection by the rCAT1 glycosylation.     

Evidence for a novel polymorphism affecting both N-linked glycosylation and ligand binding of the IgG receptor IIIB (CD16)

Immunoglobulin G Fc receptor IIIb (FcgammaRIIIb) is constitutively expressed on neutrophils, and has three allelic forms: FcgammaRIIIb-NA1, FcgammaRIIIb-NA2, and FcgammaRIIIb-SH. We identified two Japanese subjects in whom an A to G substitution at nt 221 changes asparagine (N) to serine (S) at amino acid position 45 in the FcgammaRIIIb-NA2 gene. FcgammaRIIIb-NA2-specific monoclonal antibodies (GRM1 and PEN1) did not bind to mutant neutrophils, which lack an N-linked glycosylation site. Furthermore, IgG3-mediated neutrophil phagocytosis by mutant was slightly increased as compared to wild-type donors (Note).     

Env length and N-linked glycosylation following transmission of human immunodeficiency virus Type 1 subtype B viruses

Whether there is selection for specific viral Env variants upon HIV-1 transmission is controversial. We examined the V1V2 and V1V4 regions of Env in 10 new and 8 previously described transmission pairs infected with HIV-1 subtype B, including a total of 9 pairs in which the infecting partner had developed substantial viral diversity prior to transmission. We found that during transmission of HIV-1 subtype B, as well as for other subtypes reported in the past, viral populations in recipients undergo substantial genetic bottlenecks, as well as weak evidence for a propensity to replicate viruses with shorter variable loops and fewer potential N-linked glycosylation sites.     

California serogroup Gc (G1) glycoprotein is the principal determinant of pH-dependent cell fusion and entry

Members of the California serogroup of orthobunyaviruses, particularly La Crosse (LAC) and Tahyna (TAH) viruses, are significant human pathogens in areas where their mosquito vectors are endemic. Previous studies using wild-type LAC and TAH181/57, a highly neurovirulent strain with low neuroinvasiveness (Janssen, R., Gonzalez-Scarano, F., Nathanson, N., 1984. Mechanisms of bunyavirus virulence. Comparative pathogenesis of a virulent strain of La Crosse and an avirulent strain of Tahyna virus. Lab. Invest. 50 (4), 447-455), have demonstrated that the neuroinvasive phenotype maps to the M segment, the segment that encodes the two viral glycoproteins Gn (G2) and Gc (G1), as well as a non-structural protein NSm. To further define the role of Gn and Gc in fusion and entry, we prepared a panel of recombinant M segment constructs using LAC, TAH181/57, and V22F, a monoclonal-resistant variant of LAC with deficient fusion function. These M segment constructs were then tested in two surrogate assays for virus entry: a cell-to-cell fusion assay based on T7-luciferase expression, and a pseudotype transduction assay based on the incorporation of the bunyavirus glycoproteins on an MLV backbone. Both assays demonstrated that Gc is the principal determinant of virus fusion and cell entry, and furthermore that the region delineated by amino acids 860-1442, corresponding to the membrane proximal two-thirds of Gc, is key to these processes. These results, coupled with structural modeling suggesting homologies between the carboxy region of Gc and Sindbis virus E1, suggest that the LAC Gc functions as a type II fusion protein.     

N-linked glycosylation in the CXCR4 N-terminus inhibits binding to HIV-1 envelope glycoproteins

CXCR4 is a co-receptor along with CD4 for human immunodeficiency virus type 1 (HIV-1). We investigated the role of N-linked glycosylation in the N-terminus of CXCR4 in binding to HIV-1 gp120 envelope glycoproteins. Gp120s from CXCR4 (X4) and CCR5 (R5) using HIV-1 strains bound more efficiently to non-N-glycosylated than to N-glycosylated CXCR4 proteoliposomes in a CD4-dependent manner. Similar results were observed in binding studies using non-N-glycosylated or N-glycosylated CXCR4 expressed on cells. Mutation of the N-glycosylation site N11 in CXCR4 (N11Q-CXCR4) enhanced CD4-dependent binding of X4 and R5 gp120s and allowed more efficient entry of viruses pseudotyped with X4 or R5 HIV-1 envelope glycoproteins. However, the binding of R5 gp120 to N11Q-CXCR4 and entry of R5 HIV-1 viruses into cells expressing N11Q-CXCR4 were 20- and 100- to 1000-fold less efficient, respectively, than the levels achieved using X4 gp120 or X4 HIV-1 viruses. Binding of stromal cell-derived factor (SDF)-1alpha, the natural ligand of CXCR4, and SDF-1alpha-induced signaling were reduced by the N11Q mutation. These findings demonstrate that N-glycosylation at N11 inhibits the binding of CXCR4 to X4 and R5 HIV-1 gp120, and provide a better understanding of the structural elements of CXCR4 involved in HIV-1 Env-co-receptor interactions.     

Importance of N-linked glycosylation in the functional expression of murine CD1d1

The mouse CD1d1 glycoprotein is specialized in presenting lipid antigens to a novel class of T cells called natural killer T (NKT) cells. CD1d1 is predicted to contain five potential N-linked glycosylation sites (asparagine residues at positions 25, 38, 60, 128, and 183). Glycosylation has been shown to invariably affect the molecular and functional properties of various glycoproteins, and in the current report it was found that a conservative change of the individual endogenous asparagine residues in CD1d1 to glutamine differentially affected its functional expression. Although the maturation rate of the glycosylation mutants was comparable to that of wild type, they differed in their relative levels of surface expression and in their ability to stimulate NKT cells. Mutating all five glycosylation residues resulted in the absence of detectable CD1d1 expression, with a concomitant lack of NKT cell activation. Therefore, these results demonstrate that glycosylation plays a significant role in the functional expression of CD1d1.     

Point mutations in EBV gH that abrogate or differentially affect B cell and epithelial cell fusion

Cell fusion mediated by Epstein-Barr virus requires three conserved glycoproteins, gB and gHgL, but activation is cell type specific. B cell fusion requires interaction between MHC class II and a fourth virus glycoprotein, gp42, which complexes non-covalently with gHgL. Epithelial cell fusion requires interaction between gHgL and a novel epithelial cell coreceptor and is blocked by excess gp42. We show here that gp42 interacts directly with gH and that point mutations in the region of gH recognized by an antibody that differentially inhibits epithelial and B cell fusion significantly impact both the core fusion machinery and cell-specific events. Substitution of alanine for glycine at residue 594 completely abrogates fusion with either B cells or epithelial cells. Substitution of alanine for glutamic acid at residue 595 reduces fusion with epithelial cells, greatly enhances fusion with B cells and allows low levels of B cell fusion even in the absence of gL.     

Alanine substitution of conserved residues in the cytoplasmic tail of herpes simplex virus gB can enhance or abolish cell fusion activity and viral entry

Herpes simplex virus (HSV) glycoprotein B (gB) is one of the four viral glycoproteins required for viral entry and cell fusion and is highly conserved among herpesviruses. Mutants of HSV type 2 gB were generated by substituting conserved residues in the cytoplasmic tail with alanine or by deleting 41 amino acids from the C-terminus. Some of the mutations abolished cell fusion activity and also prevented transport of gB to the cell surface, identifying residues in the gB cytoplasmic tail that are critical for intracellular transport of this glycoprotein. These mutations also prevented production of infectious virus, possibly because the mutant forms of gB were not transported to the site of envelopment. Other mutations, particularly the deletion, significantly enhanced cell fusion activity. These mutations, as well as others described previously, identify regions of the gB cytoplasmic domain that modulate cell fusion activity.