Bozue J A, Tullius M V, Wang J, Gibson B W, Munson R S
Children's Hospital Research Foundation, Ohio State University, Columbus, Ohio 43205-2696, USA.
J Biol Chem. 1999 Feb 12;274(7):4106-14. doi: 10.1074/jbc.274.7.4106.
Haemophilus ducreyi, the cause of the sexually transmitted disease chancroid produces a lipooligosaccharide (LOS) containing a terminal sialyl N-acetyllactosamine trisaccharide. Previously, we reported the identification and characterization of the N-acetylneuraminic acid cytidylsynthetase gene (neuA). Forty-nine base pairs downstream of the synthetase gene is an open reading frame (ORF) encoding a protein with a predicted molecular weight of 34,646. This protein has weak homology to the polysialyltransferase of Escherichia coli K92. Downstream of this ORF is the gene encoding the H. ducreyi homologue of the Salmonella typhimurium rmlB gene. Mutations were constructed in the neuA gene and the gene encoding the second ORF by insertion of an Omega kanamycin cassette, and isogenic strains were constructed. LOS was isolated from each strain and characterized by SDS-polyacrylamide gel electrophoresis, carbohydrate, and mass spectrometric analysis. LOS isolated from strains containing a mutation in neuA or in the second ORF, designated lst, lacked the sialic acid-containing glycoform. Complementation studies were performed. The neuA gene and the lst gene were each cloned into the shuttle vector pLS88 after polymerase chain reaction amplification. Complementation of the mutation in the lst gene was observed, but we were unable to complement the neuA mutation. Since it is possible that transcription of the neuA gene and the lst gene were coupled, we constructed a nonpolar mutation in the neuA gene. In this construct, the neuA mutation was complemented, suggesting transcriptional coupling of the neuA gene and the lst gene. Sialyltransferase activity was detected by incorporation of 14C-labeled NeuAc from CMP-NeuAc into trichloroacetic acid-precipitable material when the lst gene was overexpressed in the nonpolar neuA mutant. We conclude that the lst gene encodes the H. ducreyi sialyltransferase. Since the lst gene product has little, if any, structural relationship to other sialyltransferases, this protein represents a new class of sialyltransferase.
杜克雷嗜血杆菌是性传播疾病软下疳的病原体，它产生一种脂寡糖（LOS），其中含有末端唾液酸N-乙酰乳糖胺三糖。此前，我们报道了N-乙酰神经氨酸胞苷合成酶基因（neuA）的鉴定和特征。在合成酶基因下游49个碱基对处是一个开放阅读框（ORF），编码一种预测分子量为34,646的蛋白质。该蛋白质与大肠杆菌K92的多唾液酸转移酶有弱同源性。在这个ORF下游是编码鼠伤寒沙门氏菌rmlB基因的杜克雷嗜血杆菌同源物的基因。通过插入Omega卡那霉素盒在neuA基因和编码第二个ORF的基因中构建突变，并构建了同基因菌株。从每个菌株中分离出LOS，并通过SDS-聚丙烯酰胺凝胶电泳、碳水化合物和质谱分析进行表征。从含有neuA或第二个ORF（命名为lst）突变的菌株中分离出的LOS缺乏含唾液酸的糖型。进行了互补研究。在聚合酶链反应扩增后，将neuA基因和lst基因分别克隆到穿梭载体pLS88中。观察到lst基因突变的互补，但我们无法使neuA突变互补。由于neuA基因和lst基因的转录可能是偶联的，我们在neuA基因中构建了一个非极性突变。在这个构建体中，neuA突变得到了互补，表明neuA基因和lst基因的转录偶联。当lst基因在非极性neuA突变体中过表达时，通过将CMP-NeuAc中的14C标记NeuAc掺入三氯乙酸沉淀物质中检测到唾液酸转移酶活性。我们得出结论，lst基因编码杜克雷嗜血杆菌唾液酸转移酶。由于lst基因产物与其他唾液酸转移酶几乎没有结构关系，如果有的话，这种蛋白质代表了一种新的唾液酸转移酶类别。
