Galatrox is a C-type lectin in Bothrops atrox snake venom that selectively binds LacNAc-terminated glycans and can induce acute inflammation

Abstract

Previous studies indicate that snake venom contains glycan-binding proteins (GBPs), although the binding specificity and biological activities of many of these GBPs is unclear. Here we report our studies on the glycan binding specificity and activities of galatrox, a Bothrops atrox snake venom-derived GBP. Glycan microarray analysis indicates that galatrox binds most strongly to glycans expressing N-acetyllactosamine (LacNAc), with a significant preference for Galβ1-4GlcNAcβ over Galβ1-3GlcNAcβ compounds. Galatrox also bound immobilized laminin, a LacNAc-dense extracellular matrix component, suggesting that this GBP can bind LacNAc-bearing glycoproteins. As several endogenous mammalian GBPs utilize a similar binding LacNAc binding preference to regulate neutrophil and monocyte activity, we hypothesized that galatrox may mediate B. atrox toxicity through regulation of leukocyte activity. Indeed, galatrox bound neutrophils and promoted leukocyte chemotaxis in a carbohydrate-dependent manner. Similarly, galatrox administration into the mouse peritoneal cavity induced significant neutrophil migration and the release of pro-inflammatory cytokines IL-1α and IL-6. Exposure of bone marrow-derived macrophages to galatrox induced generation of pro-inflammatory mediators IL-6, TNF-α, and keratinocyte-derived chemokine. This signaling by galatrox was mediated via its carbohydrate recognition domain by activation of the TLR4-mediated MyD88-dependent signaling pathway. These results indicate that galatrox has pro-inflammatory activity through its interaction with LacNAc-bearing glycans on neutrophils, macrophages and extracellular matrix proteins and induce the release of pro-inflammatory mediators.

Keywords: Bothrops atrox; C-type lectin; Inflammatory response; Neutrophil migration; Snake venom.

Fig. 1.

Galatrox glycan microarray analysis. FITC-galatrox, at concentration of 0.1 µg/mL, was assessed for glycan recognition pattern using mammalian printed array version 3.2 which consists of 406 glycans in replicates of 6. The results are expressed by the mean RFU and SEM and top 15 ranked glycan structures are illustrated with its respective glycan number.

Fig. 2.

Analysis of galatrox binding to ECM components. Polystyrene microplate wells were coated with ECM components laminin and fibronectin, and BSA as random binding (n = 3 for each component and BSA). After each well was incubated for 1 h with several concentrations of FITC-galatrox (2–32 μg/mL). The analysis was performed by fluorescence reading using a Power Wave X reader on an emission wavelength of 585/20 nm. The results obtained were expressed as mean of RFU ± SEM and are representative of two independent experiments. *P < 0.05 and ***P < 0.001 compared with BSA binding.

Fig. 3.

Galatrox human neutrophil surface interaction and chemotaxis. Human neutrophils (1 × 10⁶ cells) were incubated with different concentrations of galatrox-FITC (2–32 μg/mL) for 30 min at 4°C. The CRD involvement was assessed by previous incubation of FITC-galatrox samples (8 μg/mL) with α-lactose or α-sucrose (20 mM). Cells treated with HBSS only were used as NC. After incubation, the suspension was washed with PBS and cells analyzed by flow cytometry. The treatment was performed in triplicate for each condition and the results were reported as (A) percentage ± SEM and (B) fluorescence intensity median ± SEM of galatrox-FITC⁺ neutrophil population from two independent experiments. *P < 0.05 and ***P < 0.001 compared with NC and ^###P < 0.001 when compared with galatrox 8 μg/mL preincubated with α-lactose 20 mM. (C) Human neutrophil chemotactic activity in vitro was performed using a 48-well Boyden microchamber with galatrox concentration ranging from 2 to 32 μg/mL. Also, galatrox (32 μg/mL), preincubated with 20 mM of α-lactose (Lac) or α-sucrose (Suc), was assessed for CRD involvement. NC, represented by the random migration, and fMLP (10⁻⁷ M) were used as the reference chemoattractant for PC. The cells that migrated from the upper chamber to the lower through the membrane were counted and recorded as the mean ± SEM of total neutrophil per field (C). The assay was performed in triplicate for each condition, in two independent experiments. ***P < 0.001 compared with NC. ^###P < 0.001 when comparing Galatrox 32 μg/mL which was pre-incubated or not with α-lactose 20 mM.

Fig. 4.

Mice peritoneal leukocyte migration. In vivo leukocyte migration was performed by administration of galatrox (6.25–100 μg/cavity) into male BALB/c mice peritoneum, and the cavity washed after 4 h administration. NC animals received PBS alone, and PC group carrageenan (500 μg/cavity). For time-course evaluation, mice were treated with galatrox (100 μg/cavity) and leukocyte count was evaluated 4–72 h after administration. The peritoneal wash from dose (A) and the time-course (B) assay were assessed for total and differential cell counting. The results were expressed by the mean ± SEM of leukocyte, polymorphonuclear neutrophils (PMN) and mononuclear cell (MN) concentration (cells/mL). Peritoneal wash cytokine levels from dose (C) and time-course (D) in vivo leukocyte migration were evaluated by ELISA. Each treatment group consisted of five animals (n = 5) in two independent experiments. In cell counting (A and B), *P < 0.05 and **P < 0.01 when total leukocytes groups compared with respective control. ^##P < 0.01 and ^###P < 0.001 when PMN groups compared with respective control. For cytokine level detection (C and D), *P < 0.05 for IL-1α compared with respective control, ^#P < 0.05, and ^###P < 0.001 for IL-6 compared with respective control.

Fig. 5.

BMDM-galatrox binding and cell culture inflammatory cytokine release. BMDM (1 × 10⁶ cells/mL) was incubated with different concentrations of FITC-galatrox (2–32 μg/mL) in the presence of α-lactose or α-sucrose (galatrox 16 μg/mL + 20 mM of respective sugar). Cells treated with HBSS only were used as NC (A and B). BMDM treated previously with non-conjugated galatrox (30 µg/mL) or HBSS only was incubated with conjugated anti-TLR-4 (PE), anti-TLR-2 (PE-Cy7) and anti-Dectin 1 (Alexa Fluor 647) (C and D). After incubation, the suspensions were washed with PBS and cells analyzed by flow cytometry. The results were reported as percentage ± SEM of BMDM⁺ (A and C) and fluorescence intensity median ± SEM BMDM⁺ (B and D) from two independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with control and ^###P < 0.001 when compared with galatrox 16 μg/mL preincubated with α-lactose 20 mM. Cytokines KC, IL-6 and TNF-α concentrations were determined from BMDM cell suspension isolated from wt, tlr4^−/−, tlr2^−/− and myd88^−/− animals and treated with galatrox 30 µg/mL for 24 h. Control represents cells treated with medium only. CBA system kit assay was used to detect IL-6 and TNF-α and ELISA for detection of KC production in the supernatants. Alternatively, BMDM cells isolated from wt mice were treated galatrox (30 µg/mL) preincubated or not with α-lactose (20 mM) or sucrose (20 mM), for 24 h. Culture medium supplemented or not with carbohydrates were used as controls. Cell supernatants were used to determine the TNF-α level by ELISA (G). The results were reported as mean ± SEM of cytokine concentration (pg/mL) of BDMD supernatant treated with galatrox (n = 7). ***P < 0.001 when compared with Control group and ^###P < 0.001 or ^&&&P < 0.001 when comparing wt BMDM against knockout groups and ***P < 0.001 when compared Galatrox groups with Control groups and ^###P < 0.001 when compared Galatrox sample with Galatrox sample plus α-lactose.