Abstract
The surface molecule gp82 of metacyclic trypomastigote (MT) forms of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, mediates the host cell invasion, a process critical for the establishment of infection. Gp82 is known to bind to the target cell in a receptor-dependent manner, triggering Ca2+ signal, actin cytoskeleton rearrangement and lysosome spreading. The host cell receptor for gp82 was recently identified as LAMP2, the major lysosome membrane-associated protein. To further clarify the mechanisms of MT invasion, we aimed in this study at identifying the LAMP2 domain that interacts with gp82 and investigated whether target cell PKC and ERK1/2, previously suggested to be implicated in MT invasion, are activated by gp82. Interaction of MT, or the recombinant gp82 (r-gp82), with human epithelial HeLa cells induced the activation of Ca2+-dependent PKC and ERK1/2. The LAMP2 sequence predicted to bind gp82 was mapped and the synthetic peptide based on that sequence inhibited MT invasion, impaired the binding of r-gp82 to HeLa cells, and blocked the PKC and ERK1/2 activation induced by r-gp82. Treatment of HeLa cells with specific inhibitor of focal adhesion kinase resulted in inhibition of r-gp82-induced PKC and ERK1/2 activation, as well as in alteration of the actin cytoskeleton architecture. PKC activation by r-gp82 was also impaired by treatment of HeLa cells with inhibitor of phospholipase C, which mediates the production of diacylglycerol, which activates PKC, and inositol 1,4,5-triphosphate that releases Ca2+ from intracellular stores. Taken together, our results indicate that recognition of MT gp82 by LAMP2 induces in the host cell the activation of phosholipase C, with generation of products that contribute for PKC activation and the downstream ERK1/2. This chain of events leads to the actin cytoskeleton disruption and lysosome spreading, promoting MT internalization.
Highlights
The major lysosome-associated membrane glycoproteins LAMP1 and LAMP2 are heavily glycosylated proteins, contain a single membrane-spanning segment, a major portion that resides in the luminal side of lysosomes and a short cytosolic tail (Fukuda et al, 1988; Howe et al, 1988; Granger et al, 1990) Their extensive glycosylation is apparently not necessary for normal targeting, stability, or lysosome function (Kornfeld and Mellman, 1989)
As regards LAMP2, amino acids 1-28 (Nterminal signal peptide) and 378-410 were excluded, as described on the page where the sequence was obtained and confirmed by the same online tools used for gp82. 3D model of gp82 was generated in online server Phyre2 (Protein Homology/analogY Recognition Engine V 2.0) (Kelley et al, 2015), using intensive modelling mode, and that of LAMP2 was generated in SWISS-MODEL Interactive Workspace (Waterhouse et al, 2018), without template
Our results have indicated that interaction of metacyclic trypomastigote (MT) gp82 with its host cell receptor LAMP2 induces the signaling pathway that involves the activation of PKC and the downstream ERK1/2
Summary
The major lysosome-associated membrane glycoproteins LAMP1 and LAMP2 are heavily glycosylated proteins, contain a single membrane-spanning segment, a major portion that resides in the luminal side of lysosomes and a short cytosolic tail (Fukuda et al, 1988; Howe et al, 1988; Granger et al, 1990) Their extensive glycosylation is apparently not necessary for normal targeting, stability, or lysosome function (Kornfeld and Mellman, 1989). Comparison of known lamp sequences among different species has shown that human LAMP1 has more similarity to LAMP1 from other species than to human LAMP2, and this applies to LAMP2 (Fukuda et al, 1988). LAMP1 and LAMP2 may have different functions It has been shown, for instance, that surface LAMP1, but not LAMP2, protects natural killer cells from degranulation-associated damage (Cohnen et al, 2013) and that LAMP2, but not LAMP1, plays a critical role in endosomal cholesterol transport (Schneede et al, 2011)
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