Tomana M, Zikan J, Moldoveanu Z, Kulhavy R, Bennett J C, Mestecky J
Department of Medicine, University of Alabama, Birmingham 35294.
Mol Immunol. 1993 Feb;30(3):265-75. doi: 10.1016/0161-5890(93)90055-g.
Detection of the activity of beta-1,4-galactosyltransferase (beta-1,4-GT) in suspensions of viable mouse hepatocytes, the human hepatoma cell line Hep G2, the human colonic adenocarcinoma cell line HT-29, the monocyte-like cell line U937, and human splenic B and T lymphocytes suggested the presence of beta-1,4-GT, in an enzymatically active form, on plasma membranes. The presence of beta-1,4-GT on cell surfaces was also indicated from the effect of trypsinization of live cells, which significantly reduced cell surface beta-1,4-GT activity, but did not affect the activity associated with cytoplasmic membranes. Furthermore, the presence of beta-1,4-GT on the cell surface was demonstrated by indirect immunofluorescence staining of cells with anti-beta-1,4-GT antibody. The detection of radioactivity in immunoglobulins (Ig) and their component chains after incubation with suspensions of intact cells in the presence of Mn2+ and UDP-[3H]-galactose, indicated that Ig molecules were galactosylated. In the absence of UDP-[3H]-galactose, beta-1,4-GT on cell surfaces, or immobilized on Sepharose-4B, formed stable complexes with galactose acceptors, including Ig. The efficiency of binding decreased in the order: J chain > alpha chain > mu chain > polymeric IgA2 > monomeric/polymeric IgA1 > IgM > IgG. Thus, beta-1,4-GT could act as a cell-surface receptor for Ig through a cation-dependent, lectin-like association of the beta-1,4-GT with the carbohydrate moieties of the Ig. This was confirmed by indirect surface immunofluorescence and radiolabeled ligand binding assays. The binding was inhibitable by EDTA, alpha-lactalbumin (in the presence of glucose), GlcNAc, or uridine 3',5'dialdehyde. At 37 degrees C, the apparent affinity constants and association rate constants of interaction between cell surface beta-1,4-GT on glutaraldehyde-fixed HT-29 and U937 cells and alpha 2 chain or monomeric IgA1 were in the range from 7.1 x 10(7) to 4.6 x 10(8) M-1 and from 1 x 10(5) to 3 x 10(6) M-1 s-1, respectively. The dissociation rate constants and half time of dissociation calculated from these data were in the range from 2.1 x 10(-2) to 5.0 x 10(-4) s-1 and from 33 to 1380 s, respectively. The number of alpha 2 or IgA1 molecules bound per HT-29 and U937 cell were in the range from 1.9 x 10(5) to 1.3 x 10(6). The binding of IgA by the cell surface beta-1,4-GT was not associated with internalization or the catabolic degradation of the ligand.
在活的小鼠肝细胞悬液、人肝癌细胞系Hep G2、人结肠腺癌细胞系HT - 29、单核细胞样细胞系U937以及人脾B和T淋巴细胞中检测β-1,4-半乳糖基转移酶（β-1,4-GT）的活性，结果表明在质膜上存在具有酶活性形式的β-1,4-GT。活细胞经胰蛋白酶处理后，细胞表面β-1,4-GT活性显著降低，但不影响与细胞质膜相关的活性，这也表明细胞表面存在β-1,4-GT。此外，用抗β-1,4-GT抗体对细胞进行间接免疫荧光染色，证实了细胞表面存在β-1,4-GT。在存在Mn2 +和UDP-[3H]-半乳糖的情况下，完整细胞悬液与免疫球蛋白（Ig）及其组成链孵育后检测到放射性，表明Ig分子被半乳糖基化。在没有UDP-[3H]-半乳糖的情况下，细胞表面或固定在琼脂糖-4B上的β-1,4-GT与包括Ig在内的半乳糖受体形成稳定复合物。结合效率按以下顺序降低：J链＞α链＞μ链＞聚合型IgA2＞单体/聚合型IgA1＞IgM＞IgG。因此，β-1,4-GT可通过β-1,4-GT与Ig碳水化合物部分的阳离子依赖性、凝集素样结合，作为Ig的细胞表面受体。这通过间接表面免疫荧光和放射性标记配体结合试验得到证实。该结合可被EDTA、α-乳白蛋白（在有葡萄糖存在时）、GlcNAc或尿苷3',5'-二醛抑制。在37℃时，戊二醛固定的HT - 29和U937细胞表面β-1,4-GT与α2链或单体IgA1之间相互作用的表观亲和常数和缔合速率常数分别在7.1×10(7)至4.6×10(8) M-1和1×10(5)至3×10(6) M-1 s-1范围内。根据这些数据计算出的解离速率常数和解离半衰期分别在2.1×10(-2)至5.0×10(-4) s-1和33至1380 s范围内。每个HT - 29和U937细胞结合的α2或IgA1分子数在1.9×10(5)至1.3×10(6)范围内。细胞表面β-1,4-GT对IgA的结合与配体的内化或分解代谢降解无关。
