# An Unusual Two-Domain Thyropin from Tick Saliva: NMR Solution Structure and Highly Selective Inhibition of Cysteine Cathepsins Modulated by Glycosaminoglycans

## TL;DR

This paper describes a new tick saliva protein that selectively inhibits certain human proteases and is regulated by host molecules.

## Contribution

The study introduces IrThy, a novel thyropin from ticks with unique structural and inhibitory properties.

## Key findings

- IrThy inhibits human cathepsins V, K, and L with high specificity.
- IrThy's inhibitory activity is modulated by glycosaminoglycans and pH.
- The NMR structure of a Tg1 domain reveals the reactive center responsible for inhibition.

## Abstract

The structure and biochemical properties of protease inhibitors from the thyropin family are poorly understood in parasites and pathogens. Here, we introduce a novel family member, Ir-thyropin (IrThy), which is secreted in the saliva of Ixodes ricinus ticks, vectors of Lyme borreliosis and tick-borne encephalitis. The IrThy molecule consists of two consecutive thyroglobulin type-1 (Tg1) domains with an unusual disulfide pattern. Recombinant IrThy was found to inhibit human host-derived cathepsin proteases with a high specificity for cathepsins V, K, and L among a wide range of screened cathepsins exhibiting diverse endo- and exopeptidase activities. Both Tg1 domains displayed inhibitory activities, but with distinct specificity profiles. We determined the spatial structure of one of the Tg1 domains by solution NMR spectroscopy and described its reactive center to elucidate the unique inhibitory specificity. Furthermore, we found that the inhibitory potency of IrThy was modulated in a complex manner by various glycosaminoglycans from host tissues. IrThy was additionally regulated by pH and proteolytic degradation. This study provides a comprehensive structure–function characterization of IrThy—the first investigated thyropin of parasite origin—and suggests its potential role in host–parasite interactions at the tick bite site.