Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, California 95616, USA.
Med Mycol. 2010 Aug;48(5):744-56. doi: 10.3109/13693780903496609.
Two full-length cDNAs were isolated from Coccidioides posadasii that encode two deduced proteins (CpHEX1 and CpHEX2) with homology to the glycosyl hydrolase 20 family of beta-N-acetylhexosaminidases. CpHEX1 consists of 595 amino acids, has a predicted molecular mass of 68 kDa and shares the highest identity with the N-acetylhexosaminidase (NAGA) of Aspergillus nidulans, while CpHEX2 consists of 603 amino acids, has a predicted molecular mass of 68.5 kDa and shares the highest identity with NAG1 from Paracoccidioides brasiliensis. CpHEX1 and CpHEX2 share only 23% identity and have dissimilar homologies showing more identity with other fungal beta-N-acetylhexosaminidases than with each other. Phylogenetic analysis of selected beta-N-acetylhexosaminidases placed CpHEX1 in a cluster with the orthologs from A. nidulans, Aspergillus oryzae, Penicillium chrysogenum and Candida albicans, while CpHEX2 grouped with the orthologs from P. brasiliensis and the Trichoderma spp. beta-N-acetylhexosaminidase activity and transcripts encoding CpHEX1 and CpHEX2 were detected in vitro during the spherule-endospore (SE) phase. Expression of the Cphex1 transcript exhibited a temporal increase that correlated with beta-N-acetylhexosaminidase activity, while the Cphex2 transcript remained relatively constant. The addition of N-acetylglucosamine to the cultures increased beta-N-acetylhexosaminidase activity and the expression of the Cphex1 transcript. A native beta-N-acetylhexosaminidase enzyme was purified from in vitro SE phase and identified as CpHEX1 by mass spectrometric analysis. Both the CpHEX1 and CpHEX2 cDNAs were expressed as recombinant fusion proteins and purified under denaturing conditions to apparent homogeneity but they lacked enzymatic activity.
从 Coccidioides posadasii 中分离得到两个全长 cDNA，它们编码两个具有同源性的推测蛋白（CpHEX1 和 CpHEX2），属于β-N-乙酰氨基葡萄糖苷酶 20 家族。CpHEX1 由 595 个氨基酸组成，预测分子量为 68 kDa，与 Aspergillus nidulans 的 N-乙酰氨基葡萄糖苷酶（NAGA）具有最高的同源性，而 CpHEX2 由 603 个氨基酸组成，预测分子量为 68.5 kDa，与 Paracoccidioides brasiliensis 的 NAG1 具有最高的同源性。CpHEX1 和 CpHEX2 的同源性仅为 23%，且具有不同的同源性，与其他真菌的β-N-乙酰氨基葡萄糖苷酶的同源性比对彼此的同源性更高。对选定的β-N-乙酰氨基葡萄糖苷酶的系统发育分析将 CpHEX1 置于与 A. nidulans、Aspergillus oryzae、Penicillium chrysogenum 和 Candida albicans 的同源物聚类中，而 CpHEX2 与 P. brasiliensis 和 Trichoderma spp. 的同源物聚类。在球形体-内孢子（SE）阶段的体外检测到 CpHEX1 和 CpHEX2 的β-N-乙酰氨基葡萄糖苷酶活性和编码转录本。Cphex1 转录本的表达呈时间增加，与β-N-乙酰氨基葡萄糖苷酶活性相关，而 Cphex2 转录本相对稳定。向培养物中添加 N-乙酰氨基葡萄糖会增加β-N-乙酰氨基葡萄糖苷酶活性和 Cphex1 转录本的表达。从体外 SE 相中纯化出一种天然的β-N-乙酰氨基葡萄糖苷酶酶，并通过质谱分析鉴定为 CpHEX1。CpHEX1 和 CpHEX2 cDNA 均作为重组融合蛋白表达，并在变性条件下纯化至明显均一，但缺乏酶活性。
