Analysis of multi-function of renin binding protein

肾素结合蛋白（Renin binding protein, RnBP）是从猪肾中分离出来的内源性肾素抑制剂。最近，该蛋白被鉴定为n -乙酰- d -氨基葡萄糖（GlcNAc） 2- epimase酶。该酶催化GlcNAc与n -乙酰- d -甘露糖胺（ManNAc）之间的相互转化，核苷酸是必不可少的辅助因子。我们最近的研究表明，GlcNAc 2- epimase的中间结构域参与了核苷酸的特异性和结合。为了鉴定与核苷酸结合的残基，我们在人和大鼠GlcNAc 2-外膜酶中引入了氨基酸替换。突变体的表达和特征表明，GlcNAc 2- epimase的居位171对于该酶的核苷酸结合至关重要。重组猪RnBP在缺乏核苷酸的情况下以剂量依赖的方式抑制猪肾素活性。但这种抑制作用被核苷酸如ATP、dATP、dGTP、dCTP或dTTP所中和。ATP抑制肾素与RnBP异质复合物的形成。另一方面，sh烷基化试剂n -乙基马来酰亚胺（NEM）可抑制GlcNAc - 2- epimase的活性，并伴随二聚体向该酶的单体衰变。在ATP存在的情况下，NEM没有抑制GlcNAc 2- epimase的活性。这些结果清楚地表明，核苷酸稳定了RnBP （GlcNAc 2-epimerase）的二聚体，并抑制了肾素-RnBP异质复合物HMW肾素的形成。此外，人肾素以硫氧还蛋白的融合蛋白形式在大肠杆菌细胞中表达。嵌合蛋白是积累的不溶性包涵体，在4 M胍-盐酸中溶解，并通过l -精氨酸-洗涤剂缓冲体系和系统透析进行折叠。重新折叠的融合催乳蛋白被胰蛋白酶激活。人肾素抗血清特异性抑制肾素活性。活性重组人肾素将在不久的将来用于人RnBP的生理研究。

Renin binding protein (RnBP) is an endogenous renin inhibitor originally isolated from porcine kidney. Recently the protein was identified as the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase. The enzyme catalyzes the interconversion between GlcNAc and N-acetyl-D-mannosamine (ManNAc) and nucleotide are essential as a cofactor. Our recent studies demonstrated that the middle domain of GlcNAc 2-epimerase participates in the specificity for and binding of nucleotide. To identify the residue conferring nucleotide binding, amino acid substitutions were introduced in the human and rat GlcNAc 2-epimerases. The expression and characterization of the mutants indicate that reside 171 of GlcNAc 2-epimerase is critical for the nucleotide binding of the enzyme. The recombinant porcine RnBP inhibited porcine renin activity in a dose dependent manner in the absence of nucleotide. But the inhibition was neutralized by nucleotides such as ATP,dATP,dGTP,dCTP, or dTTP. ATP inhibited the formation of hetero-complex of renin with RnBP. On the other hand, N-ethylmaleimide (NEM), a SH-alkylating reagent inhibited GlcNAc 2-epimerase activity concomitant decaying dimer to monomer of the enzyme. NEM did not inhibit GlcNAc 2-epimerase activity in the presence of ATP. These results clearly indicate that nucleotides stabilize dimeric from of RnBP (GlcNAc 2-epimerase) and inhibited the formation of renin-RnBP hetero-complex, HMW renin. Moreover, the human renin was expressed in E. coli cells as a fusion protein of thioredoxin. The chimeric protein, accumulated insoluble inclusion bodies, was solubilized in 4 M guanidine-HCl and refolded by L-arginine-detergent buffer system and by systematic dialysis. The refolded fusion prorenin was activated by trypsin. The antiserum against human kidney renin specifically inhibited the renin activity. The active recombinant human renin will be used for the physiological studies of human RnBP in near future.