Studies on Post-Translational Tyrosine Sulfation

动物细胞中的硫酸化在许多外源性和内源性化合物如苯酚、儿茶酚、类固醇衍生物等的代谢中起着重要作用。在翻译后对酪氨酸残基和碳水化合物部分的蛋白质进行修饰。这些反应是由不同的酶催化的，如硫转移酶，将3'-磷酸腺苷-5'-硫酸磷酸（PAPS）的硫酸盐部分转移到受体底物的羟基上。各种硫转移酶已被证明位于细胞的细胞质和膜细胞器中。然而，现在很清楚，在大多数情况下，翻译后酪氨酸硫酸化是由膜结合酶催化的，这些酶主要位于高尔基体中。本研究旨在研究牛肝脏中高尔基定位酪氨酸蛋白硫代转移酶，并开发一种高效的酪氨酸硫代重组蛋白体系。通过对牛肝微粒体的研究，我们发现了一种新型的膜结合的苯酚磺酰基转移酶（PST），它能催化简单苯酚衍生物、多巴胺和酪胺的磺化，并且表现出较差的热稳定性，可以归类为“单胺”形式的苯酚磺酰基转移酶。TPST在牛肝脏高尔基膜上被表征，具有对Z-Glu-Tyr、二肽和三肽以及Boc-CCK-8的活性。在所测试的底物中，在Tyr的N侧具有Asp的肽比在该位置具有Glu的肽表现出更高的反应活性。在上述位置有赖氨酸残基的肽上不能检测到硫酸化。因此，假设TPST的底物识别确实依赖于酪氨酸残基周围的氨基酸序列。水蛭素，虽然有两个Tyr残基，但只在Tyr 63位被硫酸化。而水蛭素在酵母中表达时，酪氨酸63残基未发生硫酸化。因此，有必要通过体外磺化将该重组产物转化为其天然形式。水蛭素在酪氨酸63上的磺化是用异源TPST（牛肝）实现的，酪氨酸3残基上没有磺化。这证实了牛肝TPST可以选择性地在重组蛋白中固定特定氨基酸序列上的酪氨酸残基。少

Sulfation in animal cells plays an important role in the metabolism of many exogenous and endogenous compounds such as phenol, catechol, steroid derivatives etc. And in post-translational modification of proteins on the tyrosine residue and on carbohydrate moieties. These reactions are catalyzed by distinct enzymes viz. Sulfotransferases, that transfer the sulfate moiety of 3'-Phospho-Adenosine-5'-Phosphosulfate (PAPS) to a hydroxyl group of an acceptor substrate. Various sulfotransferases have been demonstrated to be located in both the cytosol and membrane organelles of cells. However, it is now clear that post-translation tyrosine sulfation is catalyzed in most cases by membrane bound enzymes which are mostly located in the Golgi. This research was undertaken to study the Golgi located tyrosylprotein sulfotransferase in bovine liver and to develop an efficient system for tyrosine sulfation of recombinant proteins.Upon investigating the bovine liver microsomes we could identify a nov … More el membrane bound Phenol Sulfotransferase (PST) catalyzing sulfation of simple phenol derivatives, dopamine and tyramine, and showing a poor thermostability and what could be categorized as a "monoamine" form of phenol sulfotransferase.TPST has been characterized from the bovine liver Golgi membranes with activity on Z-Glu-Tyr, similar di and tri peptides and Boc-CCK-8. Among the substrates tested, peptides having a Asp on the N side of Tyr showed a higher reactivity than the peptides having a Glu at this position. Sulfation could not be detected on a peptide with a lysyl residue at the said position. Therefore it was postulated that substrate recognition of TPST, does depend on the amino acid sequence around the tyrosine residue.Hirudin, though has two Tyr residues is sulfated onl on the Tyr at position 63. However when hirudin is expressed in yeast, sulfation could not be observed on tyrosine 63 residue. Hence it is necessary to convert this recombinant product by in-vitro sulfation to its natural form. Sulfation of hirudin on the tyrosine 63 has been achieved with a heterologous TPST (bovine liver) with no sulfation on the tyrosine 3 residue. This confirmed that bovine liver TPST could selectively sulfate, tyrosine residues on specific amino acid sequences, in recombinant proteins. Less