Structure and Function of Granulocyte Antigens

粒细胞抗原的结构和功能

• 批准号: 6683846
• 负责人: David Frank Stroncek
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6683846
• 关键词: cell population study; chemical structure function; gene expression; genetic polymorphism; human genetic material tag; human tissue; immunogenetics; isoantigen; molecular cloning; neutrophil; single nucleotide polymorphism

Granulocyte antigens play an important role in cell functions including adhesion, cell activation, and binding of immunoglobulins. The purpose of these studies is to better define the molecular basis of variations in neutrophil antigens and their role in neutrophil function. Neutrophil-specific antigen HNA-2a (NB1) has been localized to CD177 glycoprotein (gp) which is expressed on subpopulations of neutrophils. PRV-1 is a gene that is over expressed in neutrophils from patients with polycythemia rubra vera. The gene encoding NB1 differs from PRV-1 at four reported nucleotides. The goal of this study was to determine if PRV-1 and NB1 were alleles of the same gene or two separate genes; moreover, if they are alleles of the same gene, to determine the gene frequencies of each allele and explore potential correlations to neutrophil CD177 gp expression. Primer pairs were used to amplify leukocyte genomic DNA in the regions surrounding the four NB1 polymorphic sites within exon 1, exon 3, exon 8, and exon 9. The four resulting amplicons were sequenced and analyzed for each donor. The size of the neutrophil population in each donor staining brightly with CD177 antibody was assessed by flow cytometry. If PRV-1 and NB1 are separate genes, then all people tested should be heterozygous for the PRV-1/NB1 polymorphisms. Since 6 of 16 donors tested were homozygous for PRV-1 polymorphisms at all 4 sites, PRV-1 and NB1 are alleles of the same gene, CD177. When the sequenced exons in the 16 donors were compared to PRV-1, 14 single nucleotid polymorphisms (SNPs) were found. Thirteen of the 14 SNPs result in amino acid changes. The G42C exon 1 NB1 polymorphisms was the most common SNP. It was found in 7 donors. The 7 SNPs in exons 1, 2 and 3 are in the CD177 gene, but it is uncertain if the 7 SNPs in exons 7, 8, and 9 are in CD177 or a homologous pseudo gene. Thirty-two SNPs were also found in introns 2, 7, and 8. Since the G42C SNP resulted in an amino acid change in the CD177 protein leader sequence the size of the CD177 bright neutrophil population was compared among donors homozygous of G at bp42, 42GG, and those homozygous for C, 42CC. The CD177 bright neutrophil population was greater in 42CC donors than in 42GG donors. These studies show that PRV-1 and NB1 are alleles of the polymorphic gene CD177. The most common SNP in bp42 predicted an amino acid change in the protein's leader sequence and effects protein expression. Future studies will be directed toward evaluating the entire CD177 gene sequence in normal donors and in patient with polycythemia vera.

粒细胞抗原在细胞功能中起重要作用，包括粘附，细胞活化和免疫球蛋白的结合。这些研究的目的是更好地定义中性粒细胞抗原变异的分子基础及其在中性粒细胞功能中的作用。中性粒细胞特异性抗原HNA-2A（NB1）已定位在CD177糖蛋白（GP）上，该糖蛋白（GP）以中性粒细胞的亚群表示。 PRV-1是一种基因，在来自多余细胞rubra Vera患者的中性粒细胞中过度表达。编码NB1的基因在四个报道的核苷酸下与PRV-1不同。这项研究的目的是确定PRV-1和NB1是否是同一基因或两个单独基因的等位基因。此外，如果它们是同一基因的等位基因，则可以确定每个等位基因的基因频率并探索与中性粒细胞CD177 GP表达的潜在相关性。使用底漆对在外显子1，外显子3，外显子8和外显子内的四个NB1多态性位点周围的区域扩增白细胞基因组DNA。对每个供体进行了测序并分析了四个所得扩增子。通过流式细胞仪评估每个供体抗体染色明亮染色的中性粒细胞种群的大小。如果PRV-1和NB1是独立的基因，则所有测试的人应构成PRV-1/NB1多态性的杂合。由于在所有4个地点的PRV-1多态性中，经过测试的16个捐赠者中有6个是纯合的，因此PRV-1和NB1是同一基因CD177的等位基因。当将16个供体的测序外显子与PRV-1进行比较时，发现了14个单核苷多态性（SNP）。 14个SNP中有13个导致氨基酸变化。 G42C外显子1 NB1多态性是最常见的SNP。它是在7个捐助者中发现的。外显子1、2和3中的7个SNP位于CD177基因中，但是不确定外显子7、8和9中的7个SNP是在CD177中还是同源伪基因。在内含子2、7和8中也发现了32个SNP。由于G42C SNP导致CD177蛋白铅序列的氨基酸变化，因此比较了BP42、42GG和CC，42GOUS的bp42 ggous g y khimozygous CD177明亮中性粒细胞的大小。 42cc供体的CD177明亮的中性粒细胞种群高于42GG供体中的CD177。这些研究表明，PRV-1和NB1是多态基因CD177的等位基因。 BP42中最常见的SNP预测蛋白质铅序列和影响蛋白质表达的氨基酸变化。未来的研究将用于评估正常供体和患有多余性无线疗法患者的整个CD177基因序列。