Recognition Of HIV Envelope gp120 by Siglec and C-type Lectin Receptors

Siglec 和 C 型凝集素受体对 HIV 包膜 gp120 的识别

• 批准号: 8946346
• 负责人: PETER D. SUN
• 金额: $ 49.06万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 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/8946346
• 关键词: Adhesions; Affinity; Antibodies; Antigen Presentation; Antigens; Binding; Biological Assay; C Type Lectin Receptors; CD209 gene; CD4 Positive T Lymphocytes; Cancer cell line; Carbohydrates; Cattle; Cell Adhesion Molecules; Cell surface; Cells; Colo205; Complex; Confocal Microscopy; Data; Dendritic Cells; Dose; Down-Regulation; Extracellular Matrix Proteins; Family; Genome; Glycoproteins; HIV; HIV Envelope Protein gp120; HIV Infections; HIV-1; Human Genome; Human Virus; Infection; Intercellular Adhesion Molecule 2; Lead; Lectin; Lectin Receptors; Ligand Binding Domain; Ligands; Lymphocyte antigen CD50; Mannans; Matrix Metalloproteinase Inhibitor; Modeling; Molecular; Molecular Conformation; Molecular Models; Mucin-1 Staining Method; Mucins; N-terminal; Oligosaccharides; Pathogenesis; Patients; Phase; Physiological; Protein Family; Proteins; Publishing; RNA Viruses; Receptor Signaling; Recombinant Proteins; Resolution; Roentgen Rays; Role; SELL gene; Scheme; Selectins; Sialic Acids; Solutions; Specificity; Structure; T memory cell; T-Cell Activation; T-Lymphocyte; Therapeutic; Translations; Tumor Markers; Viral; base; biphenyl hydrolase-related protein; carbohydrate receptor; disulfide bond; env Gene Products; extracellular; human SIGLEC5 protein; insight; member; molecular modeling; receptor; receptor binding; receptor internalization; research study; sialic acid binding Ig-like lectin; transmission process; tumor

DC-SIGN, a dendritic cell surface C-type lectin receptor, has been proposed to enhance HIV-1 infection of T cells in trans. The physiological role of DC-SIGN was thought to bind adhesion molecules, ICAM-2 and ICAM-3, to initiate a dendritic cell and T cell contact to facilitate the T cell activation and priming by dendritic cells. We expressed various extracellular truncations of DC-SIGN and DC-SIGNR as recombinant proteins and showed that 1) DC-SIGN/R forms a tetramer through the extracellular repeat region; 2) the receptor-gp120 binding affinity depends on the oligomerization of the receptor; 3) DC-SIGN/R recognition of gp120 is pH dependent such that the affinity reduced drastically under the endosomal pH condition; and 4) DC-SIGN recognizes ICAM-3 with much lower affinity than it does gp120. The structural study of DC-SIGN/R has resulted in a 1.5 E resolution crystal structure for DC-SIGN R8 and a molecular model for the tetrameric extracellular DC-SIGN/R. Based on the model, a prediction scheme was developed to evaluate potential ligands for DC-SIGN. A ligand search among the sequences of the human genome and genomes of human viruses revealed a diverse list of potential host and viral ligands of the receptor. The host ligands include not only adhesion molecules but also a family of mucin-like proteins, the extracellular matrix proteins and some tumor markers. Most of the enveloped RNA viruses appear to have ligands for the receptor. Using a bovine submaxillary mucin as the example, we showed both in solution and on the cell surface that DC-SIGN and DC-SIGNR recognize the mucin family of proteins with sub-micromolar affinity. The receptor binding to mucin competes with the receptor binding to HIV-1 gp120. Using FACS analysis and confocal microscopy, we showed that DC-SIGN preferentially recognizes MUC1-transfected cells as well as an MUC1-expressing cancer cell line Colo205. The binding of DC-SIGN/R to Colo205 cells can be blocked with mannan in a dose-dependent manner. Thus, we propose that DC-SIGN functions as an antigen- capturing receptor for both viral glycoprotein and cellular tumor and mucin-associated antigens. The receptor internalization would lead to the degradation and antigen presentation. SIGLECs (sialic-acid Ig-like binding lectins) are a family of adhesion and signaling receptors that specifically recognize sialylated carbohydrate moieties. The potential involvement of members of SIGLEC family receptors in HIV pathogenesis remain to be determined. To understand the molecular basis for the sialic acid-dependent adhesion implemented by SIGLECs and to get an insight into receptor specificity, structural studies have been carried out using two Ig-like N-terminal domains of SIGLEC-5. X-ray structure solution using molecular replacement with phased translation function uncovered unparalleled features not seen in other one-domain structures of related SIGLECs, including unusual conformation of variable loop C-C of the ligand-binding domain and a unique interdomain disulfide bond. To get an insight into receptor specificity, we have crystallized several receptor-ligand complexes using sialylated oligosaccharides commonly found at cell surfaces and in the extracellular milieu. In combination with binding assays, these structural studies have given us an insight into what governs ligand recognition and receptor specificity in SIGLEC family of lectins. In addition to Siglec receptors, we recently began to investigate the potential interaction between C-type lectin receptor, CD62L, and HIV-1 envelope protein gp120. Our preliminary data showed that gp120 binds CD62L with nM affinity both in solution and on cell surface and antibody against CD62L inhibited both gp120 binding and HIV-1 infection of CD4 T cells. Consistent with published patient data, HIV preferentially infect CD62L expressing central memory T cells and CD62L expressing nave CD4 T cells. The infection leads to down regulation and shedding of CD62L. Matrix metalloproteinase inhibitor, BB 94, inhibited HIV infection and may have potential use in therapeutics.

DC-SIGN 是一种树突状细胞表面 C 型凝集素受体，已被提议增强 HIV-1 对 T 细胞的反式感染。 DC-SIGN 的生理作用被认为是结合粘附分子 ICAM-2 和 ICAM-3，启动树突状细胞和 T 细胞接触，从而促进树突状细胞的 T 细胞激活和启动。 我们将 DC-SIGN 和 DC-SIGNR 的各种胞外截短表达为重组蛋白，并表明：1）DC-SIGN/R 通过胞外重复区域形成四聚体； 2) 受体-gp120结合亲和力取决于受体的寡聚化； 3) DC-SIGN/R对gp120的识别是pH依赖性的，因此在内体pH条件下亲和力急剧降低； 4) DC-SIGN 识别 ICAM-3 的亲和力比识别 gp120 的亲和力低得多。 DC-SIGN/R 的结构研究得出了 DC-SIGN R8 的 1.5 E 分辨率晶体结构和四聚体细胞外 DC-SIGN/R 的分子模型。 基于该模型，开发了一个预测方案来评估 DC-SIGN 的潜在配体。 对人类基因组和人类病毒基因组序列的配体搜索揭示了该受体的潜在宿主和病毒配体的多样化列表。 宿主配体不仅包括粘附分子，还包括粘蛋白样蛋白家族、细胞外基质蛋白和一些肿瘤标志物。 大多数有包膜 RNA 病毒似乎都具有受体的配体。 以牛颌下粘蛋白为例，我们在溶液中和细胞表面上均显示 DC-SIGN 和 DC-SIGNR 以亚微摩尔亲和力识别粘蛋白家族蛋白。 与粘蛋白结合的受体与与 HIV-1 gp120 结合的受体竞争。 使用FACS分析和共聚焦显微镜，我们表明DC-SIGN优先识别MUC1转染的细胞以及表达MUC1的癌细胞系Colo205。 DC-SIGN/R 与 Colo205 细胞的结合可以用甘露聚糖以剂量依赖性方式阻断。因此，我们提出 DC-SIGN 充当病毒糖蛋白和细胞肿瘤和粘蛋白相关抗原的抗原捕获受体。 受体内化会导致降解和抗原呈递。 SIGLEC（唾液酸 Ig 样结合凝集素）是特异性识别唾液酸化碳水化合物部分的粘附和信号传导受体家族。 SIGLEC 家族受体成员在 HIV 发病机制中的潜在参与仍有待确定。 为了了解 SIGLEC 实现的唾液酸依赖性粘附的分子基础并深入了解受体特异性，使用 SIGLEC-5 的两个 Ig 样 N 末端结构域进行了结构研究。使用具有定相翻译功能的分子替换的 X 射线结构解决方案揭示了相关 SIGLEC 的其他单域结构中未见的无与伦比的特征，包括配体结合域的可变环 C-C 的异常构象和独特的域间二硫键。为了深入了解受体特异性，我们使用细胞表面和细胞外环境中常见的唾液酸化寡糖结晶了几种受体-配体复合物。结合结合测定，这些结构研究使我们深入了解了 SIGLEC 凝集素家族中配体识别和受体特异性的控制因素。 除了Siglec受体之外，我们最近开始研究C型凝集素受体、CD62L和HIV-1包膜蛋白gp120之间的潜在相互作用。 我们的初步数据表明，gp120 在溶液中和细胞表面均以 nM 亲和力结合 CD62L，并且抗 CD62L 抗体抑制 gp120 结合和 CD4 T 细胞的 HIV-1 感染。与已发表的患者数据一致，HIV 优先感染表达 CD62L 的中央记忆 T 细胞和表达 CD62L 的幼稚 CD4 T 细胞。 感染导致 CD62L 下调和脱落。 基质金属蛋白酶抑制剂 BB 94 可抑制 HIV 感染，并可能在治疗中具有潜在用途。