Mialostatin, a Novel Midgut Cystatin from Ixodes ricinus Ticks: Crystal Structure and Regulation of Host Blood Digestion

Abstract

The hard tick Ixodes ricinus is a vector of Lyme disease and tick-borne encephalitis. Host blood protein digestion, essential for tick development and reproduction, occurs in tick midgut digestive cells driven by cathepsin proteases. Little is known about the regulation of the digestive proteolytic machinery of I. ricinus. Here we characterize a novel cystatin-type protease inhibitor, mialostatin, from the I. ricinus midgut. Blood feeding rapidly induced mialostatin expression in the gut, which continued after tick detachment. Recombinant mialostatin inhibited a number of I. ricinus digestive cysteine cathepsins, with the greatest potency observed against cathepsin L isoforms, with which it co-localized in midgut digestive cells. The crystal structure of mialostatin was determined at 1.55 Å to explain its unique inhibitory specificity. Finally, mialostatin effectively blocked in vitro proteolysis of blood proteins by midgut cysteine cathepsins. Mialostatin is likely to be involved in the regulation of gut-associated proteolytic pathways, making midgut cystatins promising targets for tick control strategies.

Tick cysteine protease inhibitors suppress immune responses in mannan-induced psoriasis-like inflammation

“…This study explores the therapeutic effects of four tick cystatins previously reported for their various immunomodulatory activities and specificity towards key cysteine proteases involved in different pathways related to immunity, inflammation, and homeostasis. Sialostatin L and Sialostatin L2, two cystatins isolated from the salivary glands of Ixodes scapularis ( 15 – 17 , 23 , 28 – 31 ), Iristatin, a salivary gland cystatin and Mialostatin, a midgut cystatin were isolated from Ixodes ricinus ( 18 , 19 , 21 ). Table 1 and Supplementary Figure 1 show the protease inhibitors, their primary targets, and published structures.…”

“…Mialostatin, a cystatin from the midgut of Ixodes ricinus , inhibits several digestive cysteine-cathepsins, with a high-level potency observed against cathepsin L isoforms. Mialostatin has also effectively blocked in vitro proteolysis of blood proteins by midgut cysteine cathepsins ( 19 ).…”

An evolutionary molecular adaptation of an unusual stefin from the liver fluke Fasciola hepatica redefines the cystatin superfamily

“…Well-documented classical stefins from the type 1 family and true cystatins from the type 2 family are presented, including members of human origin (stefins A and B, cystatins C, D, and F) and those from parasitic ticks (salivary sialostatins L and L2 and iristatin; salivary gland-associated Rhcyst-1; gut-associated mialostatin and OmC2). Inhibition data against various cysteine proteases, including human papain-family cathepsins L to B (hCatL to hCatB) and mammalian legumains, are presented as IC 50 (tick inhibitors ( 10 , 37 , 69 , 70 , 71 ), FhCyLS-2 data are from Table 1 ) or K i values (human inhibitors ( 4 , 72 , 73 , 74 , 75 , 76 )) and colored as a heatmap ( green scale ); n.i. means no inhibition.…”

“…Most of the examples studied originated from ticks and helminths. In ticks, it was shown that gut-associated cystatins regulate endogenous digestive proteases, while salivary cystatins that are injected into the host can inhibit the secretion of proinflammatory cytokines, reduce T-cell proliferation, or disrupt dendritic cell maturation and differentiation ( 10 , 11 ). Helminth cystatins were demonstrated to block antigen processing and presentation, interfere with the processing of pattern recognition receptors in innate immunity, modulate production of cytokines and nitric oxide, and suppress T-cell proliferation ( 12 , 13 , 14 , 15 , 16 ).…”

Tick Innate Immune Responses to Hematophagy andEhrlichiaInfection at Single-Cell Resolution

“…In ticks, cystatins have unique properties and functions primarily related to tick feeding and evasion of the host immune response. Tick cystatins are essential for the tick’s ability to feed on the host’s blood and inhibit proteases involved in host immune responses (Karim et al, 2005; Kotsyfakis et al, 2007; Parizi et al, 2020; Lu et al, 2020; Kotál et al, 2021). Similarly, ticks secrete cystatins during tick-borne pathogen infection and colonization.…”