Purification and characterization of a second immunoreactive mannoprotein from Cryptococcus neoformans that stimulates T-cell responses
Purification and characterization of a second immunoreactive mannoprotein from Cryptococcus neoformans that stimulates T-cell responses
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DOI：
10.1128/iai.70.10.5485-5493.2002
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发表时间：
2002-10-01
影响因子：
3.1
通讯作者：
Levitz, SM
中科院分区：
文献类型：
--
作者：
Huang, C;Nong, SH;Levitz, SM
虽然已知T细胞应答对于针对真菌病原体新型隐球菌的有效宿主防御是关键的，但是刺激保护性应答的抗原的特征很差，但被认为至少部分地由甘露糖蛋白组成。最近，我们建立了一组鼠CD 4（+）-T细胞杂交瘤，可以与C.新生儿抗原。纯化了刺激杂交瘤之一的甘露糖蛋白抗原MP 98，并克隆了编码MP 98的基因。在本研究中，隐球菌抗原，MP88，刺激第二个T细胞杂交瘤，X5 A3，分泌白细胞介素-2的特点。从玻璃珠破碎的C.通过阴离子交换和疏水相互作用色谱法测定新生形式。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳解析具有88 kDa的表观分子量的单一条带，并进行部分内部氨基酸测序。对MP88基因进行了克隆和序列测定。MP88具有C-末端富含丝氨酸/苏氨酸的区域，该区域可能作为广泛的O糖基化位点，随后是推定的糖基磷脂酰肌醇锚位点。搜索C。新形虫基因组数据库显示，MP88与包括MP 98在内的至少11个其他基因共享这一特征。MP88的甘露糖蛋白性质是基于（i）C.新生菌上清液刺激X5 A3和（ii）甘露糖基化配体竞争性抑制这种刺激。因此，第二隐球菌甘露糖蛋白已被确定，刺激T细胞反应，是一种疫苗的候选者。
Although T-cell responses are known to be critical for effective host defenses against the fungal pathogen Cryptococcus neoformans, the antigens that stimulate protective responses are poorly characterized but are thought to be comprised, at least in part, of mannoproteins. Recently, we created a panel of murine CD4(+)-T-cell hybridomas that react with C. neoformans antigens. A mannoprotein antigen, MP98, that stimulated one of the hybridomas was purified, and the gene encoding MP98 was cloned. In the present study, the cryptococcal antigen, MP88, that stimulated a second T-cell hybridoma, X5A3, to secrete interleukin-2 was characterized. MP88 was purified from supernatants of glass bead-disrupted C. neoformans by anion-exchange and hydrophobic interaction chromatography. A single band with an apparent molecular mass of 88 kDa was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to partial internal amino acid sequencing. The gene encoding MP88 was cloned and sequenced. MP88 features a C-terminal serine/threonine-rich region, which presumably serves as a site for extensive O glycosylation, followed by a putative glycosylphosphatidylinositol anchor site. A search of C. neoformans genomic databases revealed that MP88 shares this feature with at least 11 other genes, including MP98. The mannoprotein nature of MP88 was established based upon the capacity of (i) the mannoprotein fraction of C. neoformans supernatants to stimulate X5A3 and (ii) mannosylated ligands to competitively inhibit this stimulation. Thus, a second cryptococcal mannoprotein has been identified which stimulates T-cell responses and is a vaccine candidate.