Roles of NGlycans in Carbohydrate Mediated Cell Adhesion

项目III-N-葡聚糖在碳水化合物介导的细胞粘连中的作用(Michiko N.Fukuda，Ph.D.)α-甘露糖苷酶II(MX)是一种与高尔基体加工α-甘露糖苷酶II(MII)密切相关的酶。为了研究MX在体内的作用，我们培育了MX基因缺陷的突变小鼠，发现MX基因缺失的小鼠出生并生长正常，但MX基因缺失的雄性小鼠由于精子发生无效而不育。我们之前对HEMPAS病的研究和来自MII基因敲除小鼠的证据表明，存在一种潜在地补偿MII缺陷的酶。为了确定MX和MIi在体内是否相互补偿损失，我们建立了MIi/MX双基因敲除小鼠。MII/MX双零突变体是胚胎致死的，表明MX和MII共同对N-糖链的加工是必需的。利用这些突变的小鼠，我们将确定MX在N-糖链生物合成中的作用。通过产生条件MII/MX双零突变体，我们将确定N-聚糖对中性粒细胞和T辅助1细胞中E-和P-选择素的贡献。此外，由于我们已经在负责黄斑性角膜营养不良的磺基转移酶中发现了与L-选择素配体结构相似的产物，因此我们还将确定该磺基转移酶是否参与了L-选择素配体的合成，也将确定该磺基转移酶是否参与了小鼠体内L-选择素配体的合成。具体目的是(1)确定MX在小鼠体内N-糖链中的作用。具体目的是(1)确定MX在N-糖链生物合成中的作用，(B)确定N-糖链对E-和P-选择素配体活性的贡献，以及(3)确定小鼠I-GlcNAc6ST是否参与小鼠体内L-选择素配体的硫酸盐化。

Project III-The Roles of N-glycans in Carbohydrate-Mediated Cell Adhesion (Michiko N. Fukuda, Ph.D.) Alpha-mannosidase IIx(MX) is an enzyme closely related to the Golgi processing alpha-mannosidase II (MII). To investigate the in vivo role of MX, we generated MX-deficient ,mutant mice, and found that MX null mice were born and grew normally but MX null males are sterile due to ineffective spermatogenesis. Our previous studies on HEMPAS disease and evidence derived from the MII gene knock-out mouse suggested the existence of an enzyme potentially compensating for the MII defect. In order to determine if MX and MII compensate for each other's loss in vivo, we produced MII/MX double gene knock-out mice. MII/MX double null mutants are embryonic lethals, indicating that MX and MII together are essential for N-glycan processing. Using these mutant mice, we will define the roles of MX in N-glycan biosynthesis. By generating conditional MII/MX double null mutants, we will determine the contribution of N-glycans to E- and P-selectin in neutrophils and T helper 1 cells. In addition, because we have identified in sulfotransferase responsible for MCD (macular corneal dystrophy), whose product is structurally similar to the L-selectin ligand, we will also determine if this sulfotransferase is involved in the synthesis of L-selectin ligand, we will also determine if this sulfotransferase is involved in the synthesis of L- selectin ligand in vivo in the mouse. Specific aims are to (1) determine the role of MX in N-glycan in vivo in the mouse. Specific aims are to (1) determine the role of MX in N-glycan biosynthesis, (b) to determine the contribution of N-glycans to the ligand activities of E- and P-selectin, and (3) to determine if mouse I-GlcNAc6ST, a sulfotransferase which also functions as keratan sulfate GlcNAc6ST, is involved in sulfation of L-selectin ligand in the mouse.