Protein Tyrosine Sulfation in Animal Cells and its Simulation in Vitro in the Downstream Processing of Proteins

Fernando, P. H. Prasantha; Sakakibara, Yoichi; Abe, Shin‐ichiro; Liu, Ming‐Cheh; Nakatsu, Seiichiro; Suiko, Masahito

doi:10.1007/978-94-011-2844-5_64

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“…26 Another method is in vitro tyrosine sulfation of recombinant hirudin. 27 Pioneering proof-of-principle studies 27,28 utilized TPST enzymes extracted from a leech or bovine, thus usage of TPST on a large scale for the preparation of sulfoproteins has not been feasible. Sufficient quantities of active TPST enzymes are made possible by cloning.…”

Section: Introductionmentioning

confidence: 99%

Tyrosine phosphorylation of recombinant hirudin increases affinity to thrombin and antithrombotic activity

Volkova,

Semenyuk

2023

Proteins

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Thrombin is one of the key enzymes of the blood coagulation system and a promising target for the development of anticoagulants. One of the most specific natural thrombin inhibitors is hirudin, contained in the salivary glands of medicinal leeches. The medicinal use of recombinant hirudin is limited because of the lack of sulfation on Tyr63, resulting in a 10‐fold decrease in activity compared to native (sulfated) hirudin. In the present work, a set of hirudin derivatives was tested for affinity to thrombin: phospho‐Tyr63, Tyr63(carboxymethyl)Phe, and Tyr63Glu mutants, which mimic Tyr63 sulfation and Gln65Glu mutant and lysine‐succinylated hirudin, which enhance the overall negative charge of hirudin, as well as sulfo‐hirudin and desulfo‐hirudin as references. Using steered molecular dynamics simulations with subsequent umbrella sampling, phospho‐hirudin was shown to exhibit the highest affinity to thrombin among all hirudin analogs, including native sulfo‐hirudin; succinylated hirudin was also prospective. Phospho‐hirudin exhibited the highest antithrombotic activity in in vitro assay in human plasma. Taking into account the modern methods for obtaining phospho‐hirudin and succinylated hirudin, they are prospective as anticoagulants in clinical practice.

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