HUMAN LEUKOCYTE RECEPTORS FOR CHEMOTACTIC PEPTIDES

我们的目标是研究结构与功能之间的关系， 趋化肽C5 a和两种主要的人类白细胞受体： C5 a受体和甲酰肽受体。 这项合作研究 细胞和分子生物学家之间的合作将通过体外 诱变和随后的表达克隆的DNA序列， 原核细胞和真核细胞。 人和大鼠C5 a肽将被 在E.大肠杆菌，结晶和它们的三维结构 通过X射线衍射测定。 C5 a突变肽将通过以下方法产生： 寡核苷酸定向诱变，包括C3 a/C5 a杂合肽， 以确定C5 a的受体结合位点。 的结合特性 将测量野生型和突变的肽， 液体将通过高分辨率核磁共振进行研究。 cDNA文库将从外周血中性粒细胞mRNA制备， 来自佛波醇酯刺激的U937细胞的mRNA，并用 寡核苷酸探针，其衍生自两种的氨基酸序列 受体。 将构建表达cDNA文库，并使用 抗受体血清。 同时，将制备消减cDNA探针 来自用人DNA转染的小鼠L细胞系，其表达这些 受体。 这些探针将用于筛选cDNA文库。 的 两种受体的氨基酸序列均来源于cDNA。 将分离基因组甲酰肽受体克隆，并将外显子/内含子 基因的结构将被确定，以预测位置 肽中的功能结构域。 该基因的5'侧翼序列 包括转录起始区的序列。 合适 将检查用克隆的cDNA转染的细胞系统， 包括小鼠L-细胞、COS-猴细胞和青蛙卵母细胞。 制备的mRNA 通过用SP 6聚合酶复制cDNA的体外试验将用于 显微注射到青蛙卵母细胞中。 配体结合结构域的分析 将通过缺失扫描程序和通过 突变受体cDNA在细胞表达系统中的表达。

The goal is to study the structure-function relationship for both the chemotactic peptide C5a and two major human leukocyte receptors: the C5a-receptor and the formyl peptide receptor. This collaborative study between cellular and molecular biologists will be performed by in vitro mutagenesis and subsequent expression of cloned DNA sequences in procaryotic and eucaryotic cells. The human and rat C5a peptides will be produced in E. coli, crystallized and their three dimensional structure determined by X-ray diffraction. C5a mutant peptides will be created by oligonucleotide directed mutagenesis, including C3a/C5a hybrid peptides, in order to define the receptor binding sites of C5a. Binding properties of wild-type and mutated peptides will be measured and their conformation in liquid will be studied by high resolution nuclear magnetic resonance. cDNA libraries will be prepared from peripheral blood neutrophil mRNA and mRNA from phorbol-ester stimulated U937 cells and screened with oligonucleotide probes derived from amino acid sequences of both receptors. Expression cDNA libraries will be constructed and screened with anti-receptor sera. In parallel, subtracted cDNA probes will be prepared from mouse L-cell lines transfected with human DNA, which express these receptors. These probes will be used to screen the cDNA libraries. The amino acid sequences of both receptors will be derived from cDNA. Genomic formyl peptide receptor clones will be isolated and the exon/intron structure of the gene will be determined in order to predict the location of functional domains in the peptide. The 5' flanking sequence of the gene including the transcription start region will be determined. Suitable cellular system for transfection with cloned cDNA will be examined, including mouse L-cells, cos-monkey cells and frog oocytes. mRNA prepared in vitro by copying of cDNA with SP6 polymerase will be used for microinjection into frog oocytes. An analysis of the ligand binding domain of the receptor will be initiated by a deletion scanning procedure and by expression of mutated receptor cDNA in a cellular expression system.