Teshima G, Porter J, Yim K, Ling V, Guzzetta A
Department of Medicinal and Analytical Chemistry, Genentech, Inc., South San Francisco, California 94080.
Biochemistry. 1991 Apr 23;30(16):3916-22. doi: 10.1021/bi00230a016.
High-performance cation-exchange chromatography of recombinant soluble CD4 (rCD4) allowed the resolution of four charge variants. This charge heterogeneity could be eliminated by neuraminidase treatment of rCD4 and therefore can be attributed to different degrees of sialylation of the carbohydrate portion of this glycoprotein. A single acidic variant was observed upon cation-exchange chromatography of neuraminidase-treated rCD4 that had been stored in liquid solution, pH 7.2, at 25 degrees C for 6 months. This acidic variant was isolated by semipreparative cation-exchange chromatography and subjected to tryptic mapping analysis. Tryptic peptides were characterized by fast atom bombardment mass spectrometry (FABMS). The results of this analysis demonstrated that the acidic variant of neuraminidase-treated rCD4 is generated from deamidation at Asn-52. Digestion of the deamidated rCD4 with endoproteinase Asp-N confirmed Asn-52 as the primary site of deamidation. The ability of the deamidated rCD4 variant to bind gp120 was assessed by use of an ELISA-based binding assay. The binding capacity of the deamidated variant was 24% of the binding capacity of unmodified rCD4. The overall structure of the V1 domain in the deamidated variant was not markedly different from that of the native protein as probed with eight conformationally dependent anti-V1 monoclonal antibodies. Therefore, it appears that Asn-52 is directly involved in binding to gp120.
重组可溶性CD4（rCD4）的高效阳离子交换色谱法可分离出四种电荷变体。这种电荷异质性可通过对rCD4进行神经氨酸酶处理而消除，因此可归因于该糖蛋白碳水化合物部分不同程度的唾液酸化。对在25℃、pH 7.2的液体溶液中储存6个月的经神经氨酸酶处理的rCD4进行阳离子交换色谱分析时，观察到单一酸性变体。通过半制备阳离子交换色谱法分离出该酸性变体，并对其进行胰蛋白酶图谱分析。用快原子轰击质谱法（FABMS）对胰蛋白酶肽进行表征。该分析结果表明，经神经氨酸酶处理的rCD4的酸性变体是由Asn-52处的脱酰胺作用产生的。用天冬氨酸内肽酶对脱酰胺的rCD4进行消化，证实Asn-52是脱酰胺的主要位点。通过基于ELISA的结合试验评估脱酰胺的rCD4变体结合gp120的能力。脱酰胺变体的结合能力是未修饰rCD4结合能力 的24%。用8种构象依赖性抗V1单克隆抗体检测时，脱酰胺变体中V1结构域的整体结构与天然蛋白没有明显差异。因此，似乎Asn-52直接参与与gp120的结合。
