见于我国人群的一例St~a血型糖蛋白

在我国人群中筛查出一例疑似St~a血型糖蛋白(GP)变种。先证者为黎族男青年,属杂合子。家系调查表明,其遗传变异来自母亲。現借助限制性核酸內切酶图谱及一系列序列重叠的寡核苷酸探针,完成了其基因结构的鉴定,确证为見于我国的首例St~aGP变种。在其(δ-α)杂化基因的交叉点,δGP基因断裂于26号氨基酸残基附近,而αGP基因断裂则位于59号氨基酸残基附近。     

见于我国人群的一例MiIII血型糖蛋白

在我国人群中筛查出一例血型糖蛋白（ＧＰ）变种。先证者为海南省黎族男青年。家系调查表明,先证者及其父属纯合子,母亲为杂合子。通过免疫印迹与限制性内切酶图谱分析,确证它为见于我国的首例ＭｉＩＩＩＧＰ,属（δ－α－δ）基因杂化体。     

见于我国人群的一例MiⅢ血型糖蛋白

在我国人群中筛查出一例血型糖蛋白（ＧＰ）变种。先证者为海南省黎族男青年。家系调查表明，先证者及其父属纯合子，母亲为杂合子。通过免疫印迹与限制性内切酶图谱分析，确证它为见于我国的首例ＭｉＩＩＩＧＰ；属（δ－α－δ）基因杂化体。     

我国人红细胞膜MiⅢ血型糖蛋白的基因结构分析

在１１４名青年人中筛查出的两例血型糖蛋白（ＧＰ）变种。携有ＭｉⅢ基因特殊的变异片段。先证者均为海南省黎族男性,属杂合子。借助聚合酶链式反应（ＰＣＲ）获得跨越ＭｉⅢ基因外显子２￣４的基因组序列,再进行被扩增ＤＮＡ的直接测序,确证其基因结构属（δ－α－δ）杂化体。通过基因的微转换机理,δ基因的假外显子在其３＇端同显示５＇剪接信号的α基因外显子３及内含子３融合,前两者形成一组合外显子３．ＭｉⅢ基因的近端     

血型糖蛋白及其变种基因发生的分子机理

人红细胞膜血型糖蛋白α和δ组成ＭＮＳｓ血型系统抗原。该系统ＧＰＡ,ＧＰＢ和ＧＰＥ的基因结构已经阐明。在人群中,该系统显示了结构多态性,并具有许多α和δ基因杂化产生的变种,而不等交换与基因转换则可能是该系统进行演变和这种变种产生的分子机理。     

我国人红细胞膜MiⅢ血型糖蛋白的基因结构分析

在１１４名青年人中筛查出的两例血型糖蛋白（ＧＰ）变种,携有ＭｉⅢ基因特殊的变异片段．先证者均为海南省黎族男性,属杂合子．借助聚合酶链式反应（ＰＣＲ）获得跨越ＭｉⅢ基因外显子２～４的基因组序列,再进行被扩增ＤＮＡ的直接测序,确证其基因结构属（δ－α－δ）杂化体．通过基因的微转换机理,δ基因的假外显子在其３＇端同显示５＇剪接信号的α基因外显子３及内含子３融合,前两者形成一组合外显子３．ＭｉⅢ基因的近端（δ－α）断点（Ｂｒｅａｋｐｏｉｎｔ）在组合外显子３内（第８２５～８５０位核苷酸）;而远端（α－δ）断点则在内含子３内（第９０４～９６８位核苷酸）．     

我国青年人红细胞膜血型糖蛋白变异的筛查

采用免疫印迹技术首次调查我国人群红细胞膜血型糖蛋白(Glycophorins; GP)的变种。受检者114人,年龄17—25岁,男80名,女34名。以对GP分子不同区域具特异性的4种抗血清为探针,调查发现3个类型的10例变种,总检出率达8.8％。其中汉族的检出率为5.7％,黎族为25％,其余民族中未检出变种。4例Ⅰ型先证者红细胞膜GP的免疫印迹图谱与st~a表型相似,其中1例经血清学检查确证。1例Ⅱ型缺失δGP。其余5例Ⅲ型变种尚未见有文献报道。7例GP变种红细胞膜的唾液酸含量明显高于正常人。     

血小板膜糖蛋白(GP)Ⅲa PLA、Ⅰa807C/T 基因多态性与阿司匹林 抵抗的相关性研究

目的:探讨青岛地区汉族人群阿司匹林抵抗与血小板膜糖蛋白GPⅢaPLA、Ⅰ a807C/T基因多态性之间的关系.方法:筛选150例动脉粥样硬化患者服用阿司匹林(100mg/d)至少14d以上,根据血小板聚集功能测定将其分为阿司匹林抵抗(AR)组、阿司匹林半抵抗(ASR)组,阿司匹林敏感(AS)组.用PCR-RFLP法确定各组GPⅢaPLA、GP Ⅰa 807C/T基因型.结果:仅于ASR组检出1例PLA1/A2基因型,其余均为PLA1/A1基因型,未发现PLA2/A2基因型,差异无统计学意义(P＞0.005);GP Ⅰ a807C/T基因位点AR组、ASR组的T等位基因频率均显著高于AS组,有统计学意义(P＜0.005).结论:GPⅢaPLA2基因可能不是阿司匹林抵抗的遗传危险因素.而GP Ⅰ a 807C/T基因位点的T等位基因与阿司匹林抵抗的发生相关联,可能是阿司匹林抵抗遗传易感因素.     

一个黎族α-地中海贫血融合基因遗传家系的鉴定

鉴定海南黎族人群中发现的一种α-地中海贫血融合基因,并对其家系进行分析,探讨融合基因形成机制及遗传规律。采集先证者及其家系成员外周全血,进行血细胞分析、血红蛋白电泳和地贫常见基因型检测,并采用Gap-PCR法结合特异引物和基因测序技术对先证者基因型进行鉴定。结果显示先证者基因型为Fusion gene/-α4.2,且该融合基因是由于α珠蛋白基因的α2段与Ψα1段序列发生融合所致。家系遗传分析显示,其祖父基因型为Fusion gene/αα,伯父和父亲的基因型均为Fusion gene/-α4.2,母亲基因型为-α4.2/αwsαws,弟弟基因型为-α4.2/αwsαws。海南省黎族人群中存在有α-地贫融合基因,该发现丰富了黎族地贫基因突变数据库,对遗传咨询及地贫基因诊断和防治具有重要意义。     

趋化因子基因对HIV-1外膜蛋白基因疫苗诱导的免疫应答的影响

研究趋化因子基因对HIV-1外膜蛋白基因疫苗诱导免疫应答的影响,以探求防治HIV的新策略.将pVAX1GP120联合RANTES、MIP-1α基因或者pVAX1GP120单独免疫Balb/c小鼠,采用ELISA检测免疫小鼠的特异性抗体和IFN-γ水平,用MTT比色法检测免疫小鼠脾淋巴细胞的增殖,用乳酸脱氢酶(LDH)试验检测小鼠特异性细胞毒性T淋巴细胞(CTL)的应答.与pVAX1GP120免疫组比较,pVAX1GP120联合RANTES、MIP-1d基因免疫组小鼠血清的抗HIV-1gp120抗体滴度升高,有显著性差异(p＜0.01);与pVAX1GP120免疫组比较,pVAX1GP120联合RANTES、MIP-1d基因免疫组小鼠血清的IFN-γ升高,有显著性差异(p＜0.01);pVAX1GP120联合RANTES、MIP-1α基因免疫组小鼠的脾淋巴细胞增殖实验刺激指数(SI)以及特异性CTL活性均高于pVAX1GP120免疫组,有显著性差异(p＜0.01).RANTES、MIP-1α基因联合HIV-1外膜蛋白基因疫苗免疫小鼠,可能增强HIV特异性Th1细胞和CTL反应,RANTES、MIP-1α基因对体液免疫有加强作用.因此,RANTES、MIP- 1α基因对于HIV-1外膜蛋白基因疫苗具有较好应用前景的免疫佐剂.     

一种鉴定MNSs红细胞血型单克隆抗体类型的方法的建立

为鉴定MNSs血型单克隆细胞株6D7C9分泌的抗体类型,通过克隆、亚克隆、细胞转染等分子生物学技术建立了血型糖蛋白GPA、GPB的异源表达系统,并将其作为抗原,通过ELISA、Western 印迹法确定6D7C9分泌的McAb.结果显示,RT－PCR技术成功克隆获得了GPA、GPB血型糖蛋白编码基因,通过分别构建其重组逆转录病毒表达载体pEGZ/GPA及pEGZ/GPB,转染包装细胞293T,再感染L929细胞,经zeocin筛选2周后,RT PCR及流式细胞仪分析证实,L929/GPA和L929/GPB转基因细胞中分别有GPA、GPB目的基因的转录和蛋白表达.用稳定高表达GPA、GPB的转基因细胞通过ELISA和Western 印迹法证实单克隆细胞株6D7C9分泌的是抗GPA/GPB McAb.本研究成功地建立了血型糖蛋白GPA、GPB的异源表达系统,为MNSs血型McAb的检测及GPA、GPB蛋白的功能学研究奠定了基础.     

人红细胞膜血型糖蛋白的研究进展

血型糖蛋白(GP)是红细胞膜中主要含唾液酸的跨膜蛋白,有A、B、C和D四种.GPA是MN血型糖蛋白,GPB表达Ss、U血型,GPC、GPD则是Gerbich抗原,四种血型糖蛋白的结构有不同程度的同源性,尤以同类间的同源性程度最高,GPA在防止红细胞之间、红细胞与血管内皮细胞之间的相互作用有重要功能,并在配体诱导下影响红细胞膜的物理性质.GPC是维持红细胞正常形状、正常物理性质的重要因子.GPA和GPC的功能还分别与带3蛋白、带4.1蛋白有关.     

Evolution of the glycophorin gene family in the hominoid primates

Analysis of nucleotide sequences of the human glycophorin A (GPA) and glycophorin B (GPB) genes has indicated that the GPA gene most closely resembles the ancestral gene, whereas the GPB gene likely arose from the GPA gene by homologous recombination. To study the evolution of the glycophorin gene family in the hominoid primates, restricted DNA on Southern blots from man, pygmy chimpanzee, common chimpanzee, gorilla, orangutan, and gibbon was probed with cDNA fragments encoding the human GPA and GPB coding and 3-untranslated regions. This showed the presence in all of the hominoid primates of at least one GPA-like gene. In addition, at least one GPB-like gene was detected in man, both chimpanzee species, and gorilla, strongly suggesting that the event that produced the GPB gene occurred in the common ancestor of man-chimpanzee-gorilla. An unexpected finding in this study was the conservation ofEcoRI restriction sites relative to those of the other four enzymes used; the significance of this observation is unclear, but raises the question of nonrandomness ofEcoRI restriction sites in noncoding regions. Further analysis of the evolution of this multigene family, including nucleotide sequence analysis, will be useful in clarification of the evolutionary relationships of the hominoid primates, in correlation with the structure and function of the glycophorin molecules, and in assessment of the role of evolution in the autogenicity of glycophorin determinants.This work was supported in part by National Institutes of Health Grants AM33463 and CA33000.     

Distribution of glycophorin on the surface of human erythrocyte membranes and its association with intramembrane particles: An immunochemical and freeze-fracture study of normal and En(a−) erythrocytes

Human erythrocyte membranes of the En(a–) blood group lack the major sialoglycoprotein (glycophorin). By absorption of a crude antiglycophorin antiserum with En(a–) membranes a specific antiglycophorin antiserum was obtained. By immune electron microscopy we showed that glycophorin is randomly distributed on the surface of normal erythrocytes. When polycationized ferritin, which mainly binds to glycophorin, was used as a marker a similar even labeling of normal erythrocyte membranes was seen. En(a–) membranes bound much less of this marker. In freeze-fracturing the intramembrane particles of both membrane types had a similar distribution and appeared in equal amounts. However, partial removal of spectrin from these membranes, followed by incubation at pH 6 resulted in more extensive aggregation of the particles in En(a–) membranes than in normal membranes. The results may be interpreted as glycophorin contributing by electrostatic repulsion to the random distribution of the intramembrane particles in normal cells. This repulsion is weakened in En(a–) cells by the lack of glycophorin.     

'Wave-type' structure of a synthetic hexaglycosylated decapeptide: A part of the extracellular domain of human glycophorin A

The three-dimensional structure of a glycopeptide, His-Thr*-Ser*-Thr*-Ser*-Ser*-Ser*-Val-Thr-Lys, with 2-acetamido-2-deoxy--D-galactose (GalNAc) residues linked to six adjacent amino acids from Thr-10 to Ser-15, was studied by NMR spectroscopy and molecular dynamics (MD) simulations. The hexaglycosylated decapeptide is part of the extracellular domain of human glycophorin A and shows an extended structure of the peptide backbone due to O-glycosylation. Furthermore, each GalNAc residue exhibits one and only one NOE contact from the NHAc proton to the backbone amide proton of the amino acid that the sugar is directly bound to. This indicates a strong preference for the orientation of all GalNAc residues towards the N-terminus. NOE build-up curves were used to determine 42 inter-proton distances that, in connection with angles of the peptide backbone obtained from 3J-coupling constants, resulted in constraints for a MD simulation in water. The NMR data and the MD simulations show a preference for an extended backbone structure. The GalNAc residues are located alternatingly on opposite sides of the backbone and reduce the flexibility of the peptide backbone. The conformation of the molecule is relatively rigid and shows a 'wave-type' 3D structure of the peptide backbone within the glycosylation cluster. This new structural element is also supported by the unusual CD spectrum of the glycopeptide.     

Magnetic resonance study of 13C-enriched glycophorin A reconstituted into phospholipid vesicles

$\text{[S-[}{}^{13}\text{C}\text{]}\text{methylmethionine-8 and -81]glycophorin}$ A was reconstituted into l-α-phosphatidyl choline vesicles. Results indicate that the $\text{S-[}{}^{13}\text{C}\text{]}\text{methylmethyionine}\text{-81}$ residue in the phospholipid bilayer has limited mobility and is not susceptible to dealkylation, whereas the opposite effects are indicated for the $\text{S-[}{}^{13}\text{C}\text{]}\text{methylmethionine}\text{-8}$ residue.     

Direct modification of the glycocalyx of a cultured muscle cell line by incorporation of foreign gangliosides and an integral membrane glycoprotein

As part of a program to better understand the cause-or-effect nature of the relationship between cell surface carbohydrate and cell properties and behaviour, experiments have been carried out on direct modification of the glycocalyx of cultured cells. Modification was by incorporation of gangliosides and an integral membrane glycoprotein chosen to be dissimilar to species occurring naturally in the cell line. Two methods of incorporation were investigated: simple addition of the new components to the culture medium for various times, or assembly of the components into the walls of lipid vesicles which were subsequently fused with cells. Gangliosides from beef brain and glycophorin, the major human erythrocyte sialoglycoprotein, were successfully added to the surface of myoblasts in quantities sufficient to represent a significant perturbation. Changes in cell adhesion, morphology, and viability were observed which seem to be a direct result of glycocalyx modification.     

A monoclonal antibody to swine erythrocytes recognizes the B blood group on the major glycophorin

Recently monoclonal antibodies (mAbs) to swine red blood cells have been described. One of them (1AC11) was specific for the major swine glycoprotein with a molecular weight of 45 kDa and another mAb, 2G2, recognized the B ^a allele in the B system of swine blood groups. Immunoblotting experiments to characterize the mAb 2G2 indicated that it reacts with an antigen of 45 kDa, present on the aqueous phase, glycophorin fraction, of swine red blood cells with the B ^a allele and does not react with B ^bB^b homozygous cells. The antigen recognized by 2G2 has the same characteristics as the major glycophorin recognized by 1AC11, so we can conclude that the B system of the swine blood group is on the major glycophorin of swine erythrocyte membranes.     

Hsa, an adhesin of Streptococcus gordonii DL1, binds to α2-3-linked sialic acid on glycophorin A of the erythrocyte membrane

Bacterial recognition of host sialic acid-containing receptors plays an important role in microbial colonization of the human oral cavity. The aggregation of human platelets by Streptococcus gordonii DL1 is implicated in the pathogenesis of infective endocarditis. In addition, we consider that hemagglutination of this organism may act as an additive factor to increase the severity of this disease. We previously reported that this interaction requires the bacterial expression of a 203-kDa protein (Hsa), which has sialic acid-binding activity. In the present study, we confirmed that erythrocyte surface sialoglycoproteins are the receptors for Hsa. We examined the effects of proteinase K, chymotrypsin, phospholipase C, and α(2-3) or α(2-3, 6, 8) neuraminidase on hemagglutination activity and found that the interaction occurs between Hsa and α2-3-linked sialic acid-containing proteins of erythrocytes. We expressed recombinant NR2, which is the putative binding domain of Hsa, fused with GST in Escherichia coli BL21. Dot-blot analysis demonstrated that GST-HsaNR2 binds both glycophorin A (GPA) and band 3. Moreover, GPA and a small amount of band 3 were detected by GST pull-down assays. These findings indicate that S. gordonii Hsa specifically binds to GPA and band 3, α2-3-linked sialic acid membrane glycoproteins.