Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv 79005, Ukraine.
J Mol Recognit. 2011 Jul-Aug;24(4):576-84. doi: 10.1002/jmr.1071. Epub 2010 Nov 16.
Cell surface sialylation is known to be tightly connected with tumorigenicity, invasiveness, metastatic potential, clearance of aged cells, while the sialylation of IgG molecules determines their anti-inflammatory properties. Four sialidases - hydrolytic enzymes responsible for cleavage of sialic residues - were described in different cellular compartments. However, sialidases activity in body fluids, and specifically in blood serum, remains poorly studied. Here, we characterize first known IgG antibodies possessing sialidase-like activity in blood serum of multiple myeloma (MM) patients. Ig fractions were precipitated with ammonium sulfate (50% of saturation) from blood serum of 12 healthy donors and 14 MM patients, and screened for the presence of sialidase activity by using 4-MUNA (2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid) as substrate. High level of sialidase activity was detected in the MM patients, but not in healthy donors. Subsequent antibody purification by protein-G affinity chromatography and HPLC size exclusion chromatography at acidic conditions demonstrated that sialidase activity was attributable to IgG molecules. Sialidase activity was also specific for (Fab)(2) fragment of IgG and blocked by sialidase inhibitor DANA. Sialidase activity of IgG molecule was also confirmed by in gel assay for cleavage of sialidase substrate. Kinetic parameters of the catalysis reaction were described by Michaelis-Menten equation with K(m) = 44.4-108 µM and k(cat) = 2.7-23.1 min(-1). The action of IgG possessing sialidase-like activity towards human red blood cells resulted in a subsequent increase in their agglutination by the peanut agglutinin, that confirms their desialylation by the studied IgG. This is the first demonstration of the intrinsic sialidase activity of IgG isolated from blood serum of MM patients.
细胞表面的唾液酸化与致瘤性、侵袭性、转移性、衰老细胞的清除密切相关，而 IgG 分子的唾液酸化决定了它们的抗炎特性。四种唾液酸酶——负责切割唾液酸残基的水解酶——在不同的细胞区室中被描述。然而，体液中，特别是血清中的唾液酸酶活性仍研究甚少。在这里，我们描述了首次在多发性骨髓瘤（MM）患者血清中发现具有唾液酸酶样活性的 IgG 抗体。从 12 名健康供体和 14 名 MM 患者的血清中用硫酸铵（饱和度为 50%）沉淀 Ig 部分，并使用 4-MUNA（2'-(4-甲基伞形酮基)-α-D-N-乙酰神经氨酸）作为底物筛选是否存在唾液酸酶活性。在 MM 患者中检测到高水平的唾液酸酶活性，但在健康供体中未检测到。随后通过蛋白 G 亲和层析和酸性条件下的 HPLC 分子筛层析对抗体进行纯化，证明唾液酸酶活性归因于 IgG 分子。唾液酸酶活性也特异性针对 IgG 的（Fab）2 片段，并被唾液酸酶抑制剂 DANA 阻断。通过凝胶内分析证实了 IgG 分子的唾液酸酶活性，用于切割唾液酸酶底物。催化反应的动力学参数通过米氏方程描述，Km 值为 44.4-108 μM，kcat 值为 2.7-23.1 min-1。具有唾液酸酶样活性的 IgG 对人红细胞的作用导致随后花生凝集素对其凝集增加，这证实了所研究的 IgG 对其去唾液酸化。这是首次从 MM 患者血清中分离的 IgG 具有内在唾液酸酶活性的证明。
