Identification of Escherichia coli outer membrane protein A receptor on human brain microvascular endothelial cells
Identification of Escherichia coli outer membrane protein A receptor on human brain microvascular endothelial cells
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DOI：
10.1128/iai.70.8.4556-4563.2002
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发表时间：
2002-08-01
影响因子：
3.1
通讯作者：
Prasadarao, NV
中科院分区：
文献类型：
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作者：
Prasadarao, NV
尽管有活性抗生素可用，新生儿大肠埃希氏菌脑膜炎仍是一个诊断和治疗挑战。我们早期的研究表明，外膜蛋白A(OmpA)是大肠杆菌穿越血脑屏障的主要因素之一，血脑屏障构成了脑微血管内皮细胞(BMEC)的衬里。在这项研究中，我们发现OmpA与95 kDa的人BMEC(HBMEC)糖蛋白(ECGP)结合，以促进大肠杆菌的侵袭。小麦胚芽凝集素和山毛榉凝集素(MAL)亲和层析部分纯化了ECGP。MAL亲和纯化的HBMEC蛋白可与OmpA(+)大肠杆菌结合，但不能与OmpA(-)大肠杆菌结合。此外，去糖基化的MAL结合蛋白仍然与OmpA(+)大肠杆菌相互作用，表明蛋白质骨架在介导OmpA与HBMEC结合中的作用。有趣的是，MAIL亲和力结合的部分显示了另外一个蛋白质，一个65 kDa的蛋白质，除了ECGP外，它还与OmpA(+)大肠杆菌结合。此外，65 kDa的蛋白被证明是ECCP的切割产物。抗ECGP抗体对感染OmpA(+)大肠埃希菌的HBMEC的免疫细胞化学研究表明，ECGP聚集在大肠杆菌的进入部位。在人脑组织切片上，抗ECGP抗体也能与微血管内皮细胞反应，表明ECGP在大肠杆菌脑膜炎中具有生物学意义。ECGP的部分N端氨基酸序列与gp96有87%的序列同源性，gp96是一种经常在细胞表面表达的内质网驻留分子伴侣。相反，65 kDa的蛋白可能是ECGP的内部部分，与先前报道的S菌毛结合唾液糖蛋白的序列同源性为70%。这些结果表明，OmpA通过碳水化合物表位与ECGP相互作用，并与侵袭HBMEC的蛋白质部分相互作用。
Neonatal Escherichia coli meningitis continues to be a diagnostic and treatment challenge despite the availability of active antibiotics. Our earlier studies have shown that outer membrane protein A (OmpA) is one of the major factors responsible for Escherichia coli traversal across the blood-brain barrier that constitutes a lining of brain microvascular endothelial cells (BMEC). In this study we showed that OmpA binds to a 95-kDa human BMEC (HBMEC) glycoprotein (Ecgp) for E. coli invasion. Ecgp was partially purified by wheat germ agglutinin and Maackia amurensis lectin (MAL) affinity chromatography. The MAL affinity-purified HBMEC proteins bound to OmpA(+) E. coli but not to OmpA(-) E. coli. In addition, the deglycosylated MAL-bound proteins still interact with OmpA(+) E. coli, indicating the role of protein backbone in mediating the OmpA binding to HBMEC. Interestingly, the MAIL affinity-bound fraction showed one more protein, a 65-kDa protein that bound to OmpA(+) E. coli in addition to Ecgp. Further, the 65-kDa protein was shown to be a cleavage product of Ecgp. Immunocytochemistry of HBMEC infected with OmpA(+) E. coli by using anti-Ecgp antibody suggests that Ecgp clusters at the E. coli entry site. Anti-Ecgp antibody also reacted to microvascular endothelium on human brain tissue sections, indicating the biological relevance of Ecgp in E. coli meningitis. Partial N-terminal amino acid sequence of Ecgp suggested that it has 87% sequence homology to gp96, an endoplasmic reticulum-resident molecular chaperone that is often expressed on the cell surface. In contrast, the 65-kDa protein, which could be the internal portion of Ecgp, showed 70% sequence homology to an S-fimbria-binding sialoglycoprotein reported earlier. These results suggest that OmpA interacts with Ecgp via the carbohydrate epitope, as well as with the protein portion for invading HBMEC.