Hemoglobin Digestion in Blood-Feeding Ticks: Mapping a Multipeptidase Pathway by Functional Proteomics

Abstract

SUMMARY Hemoglobin digestion is an essential process for blood-feeding parasites. Using chemical tools, we deconvoluted the intracellular hemoglobinolytic cascade in the tick Ixodes ricinus, a vector of Lyme disease and tick-borne encephalitis. In tick gut tissue, a network of peptidases was demonstrated through imaging with specific activity-based probes and activity profiling with peptidic substrates/inhibitors. This peptidase network is induced upon blood feeding and degrades hemoglobin at acidic pH. Selective inhibitors were applied to dissect the roles of the individual peptidases and determine the peptidase-specific cleavage map of the hemoglobin molecule. The degradation pathway is initiated by endopeptidases of aspartic and cysteine class (cathepsin D supported by cathepsin L and legumain) and continued by cysteine amino- and carboxy-dipeptidases (cathepsins C and B). The identified enzymes are potential targets to developing novel anti-tick vaccines.

Ancestral reconstruction of tick lineages

“…Hemoglobin is specifically taken up via receptor-mediated endocytosis and targeted to endosomes for digestion before heme is targeted to the hemosome (Coons et al 1986;Lara et al 2005;Sojka et al 2013). In ixodids, digestion occurs via a sequential cascade of proteolytic enzymes that include aspartic (cathepsin D), cysteine (cathepsin B, C and L) and legumain-type asparaginyl peptidases that show differential expression patterns during feeding (Franta et al 2010;Horn et al 2009;Sojka et al 2008). These peptidases seem to be conserved in prostriates and metastriates (Mans, 2014 .…”

Hlcyst-1 and Hlcyst-2 are Potential Inhibitors of HlCPL-A in the Midgut of the Ixodid Tick Haemaphysalis longicornis

“…Midgut proteolytic enzymes including aspartic protease (longepsin) [7], serine carboxypeptidase (HlSCP) [17] and legumain (HlLgm) [5] have been ascribed to play roles in the degradation of Hb to peptides in the Haemaphysalis longicornis. Accordingly, the present data and those available for other tick species suggest that an enzyme cascade for the proteolysis of Hb operates in the midgut of the ticks [10].Cysteine proteases comprise a group of proteolytic enzymes that cleave peptide bonds by use of a reactive cysteine residue at the catalytic site. One of the families, C1 includes the lysosomal cathepsins B, H, and L, which play a major role in cell protein turnover following their release from the lysosomes in various pathological processes such as inflammation and tumor invasion [23].…”

Localization and function ofRhipicephalus (Boophilus) microplusvitellin-degrading cysteine endopeptidase

“…Biochemical screenings demonstrate that multiple similar activities are present in R. microplus (Mendiola et al 1996) and Ixodes ricinus (Sojka et al 2008;Horn et al 2009). So we cannot dismiss the possibility that the antibody used here can recognize isoforms of cysteine endopeptidase.…”