Synthesis, isolation, and characterization of endogenous beta-galactoside-binding lectins in human leukocytes.
Synthesis, isolation, and characterization of endogenous beta-galactoside-binding lectins in human leukocytes.
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人白细胞内源性 β-半乳糖苷结合凝集素的合成、分离和表征。
作者：
Allen,HJ;Gottstine,S;Sharma,A;DiCioccio,RA;Swank,RT;Li,H
Roswell Park Cancer Institute，布法罗，New York 14263外科肿瘤学、妇科肿瘤学、细胞和分子生物学系，Canisius College，布法罗，纽约14208生物学系，1991年4月25日接收;修订版手册，1991年6月28日接收摘要：半乳糖凝集素是一种β-半乳糖苷结合凝集素，通过亲和层析从人血沉棕黄层细胞（外周血白细胞）和脾脏中分离得到。天然血沉棕黄层半乳糖肽的分子量（32 K）与脾半乳糖肽的分子量相似。它们的亚基分子量（14.5 K）、p/（4.60-4.85）和氨基酸组成相同。两种galaptin显示出一个单一的多肽的存在下，当进行反相HPLC。针对脾半乳糖肽14.5-kDa亚基的单特异性兔多克隆抗血清与血沉棕黄层细胞、EB病毒永生化B淋巴母细胞和HL-60早幼粒细胞白血病细胞中存在的14.5-kDa多肽反应。然而，半乳糖苷肽不能在体外由血沉棕黄层细胞合成。相反，合成了Mt 15.5-16.5 K的单体β-半乳糖苷结合蛋白，其在免疫学上不同于半乳糖肽。这种半乳糖苷结合蛋白可通过聚丙烯酰胺凝胶电泳和阴离子交换色谱法与半乳糖肽分离。相反，免疫沉淀实验证实半乳糖肽由B淋巴母细胞样细胞合成。用聚合酶链反应（PCR）扩增出编码galaptin的B类淋巴母细胞mRNA的cDNA。对扩增产物进行部分序列测定，共鉴定出299个核苷酸。衍生的氨基酸对应于发现存在于人脾半乳糖肽中的残基6-65、84-114和118-126。免疫组织化学分析显示，galaptin分布在整个细胞质的B淋巴母细胞样细胞，而不是本地化的细胞表面。这里呈现的结果表明半乳糖肽存在于多种白细胞中，包括血沉棕黄层细胞。虽然血沉棕黄层细胞可以积累半乳糖肽，但它们在体外不合成半乳糖肽。合成的15.5-16.5-kDa/β-半乳糖苷结合凝集素似乎不在血沉棕黄层细胞中积累，并且其可能是分泌蛋白[艾伦，H. J.，等人（1986）Immunol.Invest. 15，123-138]。e S型凝集素（Drickamer，1989）是可溶性的、通常不依赖于阳离子的、巯基依赖性蛋白质，并且被发现存在于
Departments of Surgical Oncology, Gynecologic Oncology, and Cell and Molecular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263, and Biology Department, CanisiusCollege, Buffalo, New York 14208 Received April 25, 1991; Revised Manuscript Received June 28, 1991 abstract: Galaptin, a/3-galactoside-binding lectin, was isolated from human buffy coat cells (peripheral leukocytes) and spleen by affinity chromatography. The molecular weight (32K) of the native buffy coat galaptin was similar to that for splenic galaptin. Their subunit molecular weight (14.5 K), p/(4.60-4.85), and amino acid composition were identical. Both galaptins showed the presence of a single polypeptide when subjected to reversed-phase HPLC. Monospecific rabbit polyclonalantiserum raised against the 14.5-kDa subunit of splenic galaptin reacted with a 14.5-kDa polypeptide present in buffy coat cells, Epstein-Barr virus-immortalized B lymphoblastoid cells, and HL-60 promyelocytic leukemia cells. However, galaptin was not synthesized in vitro by buffy coat cells. Rather, a monomeric/3-galactoside-binding protein of Mt 15.5-16.5 K that is immunologically distinct from galaptin was synthesized. This galactoside-binding protein was separable from galaptin by polyacrylamide gel electrophoresis and by anion-exchange chro-matography. In contrast, immunoprecipitation experiments confirmed that galaptin was synthesized by the B lymphoblastoid cells. cDNA corresponding to the B lymphoblastoid cell mRNA encoding galaptin was amplified by thepolymerase chain reaction. The amplified product was partially sequenced, and 299 nucleotides were identified. The derived aminoacids corresponded to residues 6-65, 84—114, and 118-126 found to be present in human splenic galaptin. Immunohistochemical analyses revealed that galaptin was distributed throughout the cytoplasm of B lymphoblastoid cells rather than being localized to the cell surface. The results presented here demonstrate that galaptin is present in a variety of leukocytes including buffy coat cells. Although buffy coat cells may accumulate galaptin, they do not synthesize it in vitro. The 15.5-16.5-kDa/3-galactoside-binding lectin that is synthesized does not appear to accumulate in the buffy coat cells, and it may be a secretory protein [Allen, H. J., et al.(1986) Immunol. Invest. 15, 123-138]. e S-type lectins (Drickamer, 1989) are soluble, generally cation-independent, thiol-dependent proteins and are found