Circulating Biomphalaria glabrata hemocyte subpopulations possess shared schistosome glycans and receptors capable of binding larval glycoconjugates

Abstract

Host lectin-like recognition molecules may play an important role in innate resistance in Biomphalaria glabrata snails to larval schistosome infection, thus implicating parasite-expressed glycans as putative ligands for these lectin receptors. While host lectins may utilize specific glycan structures for parasite recognition, it also has been hypothesized that the parasite may use this system to evade immune detection by mimicking naturally-expressed host glycans, resulting in reduced immunorecognition capacity. By employing immunocytochemical (ICC) and Western blot assays using schistosome glycan-specific monoclonal antibodies (mABs) we sought to identify specific glycan epitopes (glycotopes) shared in common between larval Schistosoma mansoni and B. glabrata hemocytes, the primary immune effector cells in snails. Results confirmed the presence of selected larval glycotopes on subpopulations of hemocytes by ICC and association with numerous hemocyte proteins by Western blot analyses, including a trimannosyl core N-glycan (TriMan), and two fucosylated lacdiNAc (LDN) variants, F-LDN and F-LDN-F. Snail strain differences were seen in the prevalence of constitutively expressed F-LDN on hemocytes, and in the patterns of protein immunoreactivity with these mABs. In contrast, there was little to no hemocyte reactivity with mABs for Lewis X (LeX), LDN, LDN-F or LDN-DF. When intact hemocytes were exposed to larval transformation products (LTPs), distinct cell subpopulations displayed weak (LeX, LDN-DF) to moderate (LDN, LDN-F) glycotope reactivity by ICC, including snail strain differences in the prevalence of LDN-reactive cellular subsets. Far-Western blot analyses of the hemocytes following exposure to larval transformation proteins (LTPs) also revealed multiple mAB-reactive hemocyte protein bands for LeX, LDN, LDN-F, and LDN-DF. These results demonstrate the existence of complex patterns of shared larval glycan constitutively expressed on hemocytes and their proteins, as well as the ability of hemocytes to acquire shared glycans by the selective binding of parasite-released LTP. Unraveling the functional significance of these naturally expressed and acquired shared glycans on specific hemocyte populations represents an important challenge for future investigations.

Figure 1. “Normalized” hemocyte protein samples from NMRI and BS-90 Biomphalaria glabrata snail strains

Prior to Western/far-Western blot analyses of shared glycotopes, single populations of pooled hemocytes were isolated from the NMRI (N) and BS-90 (B) strains of B. glabrata, subjected to SDS-PAGE, and stained with Coomassie brilliant blue or silver. Based on initial staining intensities, hemocyte samples were adjusted so NMRI and BS-90 snails exhibited similar intensities of protein banding indicating comparable sample loading between strains. Molecular weight markers are indicated on the right.

Figure 2. Immunocytochemical (ICC) and Western (−LTP)/far-Western (+LTP) blot analyses of hemocyte-associated glycotopes recognized by mABs binding to (A) trimannosyl core N-glycan [TriMan; Man(α1–6)[Man α1–3)]Man(β1–4)GlcNAc(β1–4)GlcNAc], and the fucosylated lacdiNAc variants (B) F-LDN-F; Fuc(α1–3)GalNAc(β1–4)[Fuc(α1–3)]GlcNAc and (C) F-LDN; Fuc[(α1–3)]GalNAc(β1–4)GlcNAc

The ICC images of hemocytes (right panel) showing the heterogeneity of staining intensities and cell-types exhibiting specific glycotope reactivities within a single hemocyte population of both B. glabrata snail strains. TriMan-associated glycans are distributed mainly on granulocyte-type cells and substrate-adherent plasma proteins, although small spread cells (Fig. 2A; arrows) and distinctive rounded hemocytes (type “B” hyalinocyte [Schoenberg and Cheng, 1980; Yoshino and Granath, 1994]; Fig. 2A, arrowheads) completely lack TriMan glycotope expression. In contrast, granulocytes staining with F-LDN and F-LDN-F mABs were highly variable, ranging in reactivities from intensely fluorescent to negative (Figs. 2B, 2C; arrows). Moreover, the TriMan-negative type “B” hyalinocytes exhibited strong F-LDN-F mAB staining (Fig. 2B; arrowheads). Hemocyte treatment with LTPs had no effect on the prevalence or cellular distribution of glycotopes in either snail strain. Western blot analyses of shared glycotopes associated with hemocyte proteins (–LTP; left blot panels) showed a similar banding profile for TriMan glycotopes between NMRI (N) and BS-90 (B) snail strains, with the exception of bands between 15–20 kDa (Fig. 2A; arrows). A greater intensity and distribution of F-LDN-F (Fig. B) and F-LDN (Fig. C), however, was noted for N B. glabrata hemocyte proteins compared to the B snail strain. Using a far-Western blot approach, additional F-LDN-F and F-LDN-reactive bands (15–37 kDa range) were seen in blots exposed to LTP (“+LTP”, right blot panels) prior to specific mAB exposure. Arrows in Figs. 2B and 2C (“+LTP” panels) indicate appearance of reactive bands unique to the B snail strain.

Figure 3. Immunocytochemical (ICC) and Western (−LTP)/far-Western (+LTP) blot analyses of hemocyte-associated glycotopes recognized by mABs generated to schistosome lacdiNAc (LDN; GalNAc(β1–4)GlcNAc) (Fig. 3A) and LDN-F (GalNAc(β1–4)[Fuc(α1–3)]GlcNAc) (Fig. 3B)

Untreated intact hemocytes display little to no anti-LDN or anti-LDN-F mAB reactivity in both ICC (−LTP; right ICC panel) and corresponding Western blot (−LTP; left blot panel) analyses of naturally shared LDN and LDN-F glycotopes. However, exposure of intact cells or blotted hemocyte proteins to LTP resulted in the appearance of glycotope-positive hemocyte subpopulations (+LTP; right ICC panel) and immunoreactive bands in far-Western blots (+LTP; right blot panel). Arrows indicate prominent NMRI (N) and BS-90 (B) hemocyte protein bands displaying both LDN and LDN-F glycotopes in LTP-treated blots.

Figure 4. Immunocytochemical (ICC) and Western (−LTP)/far-Western (+LTP) blot analyses of hemocyte-associated glycotopes recognized by mABs generated to schistosome LDN-DF (GalNAc(β1–4)[Fuc(α1–2)Fuc(α1–3)]GlcNAc) (Fig. 4A) and Lewis × (LeX; Galβ1–4[Fuc(α1–3)]GlcNAc) (Fig. 4B)

Using ICC (−LTP; right ICC panel) and Western blot (−LTP; left blot panel) analyses intact hemocytes and their blotted proteins exhibited no reactivity to LDN-DF and LeX mABs. By contrast, small subsets of intact hemocytes (arrows; +LTP; right ICC panel) and a range of cellular proteins (+LTP; right blot panel) became immunopositive following LTP treatment. Discrete groups of hemocyte proteins appear to bind both LDN-DF- and LeX-bearing LTP glycoconjugates in NMRI (N) and BS-90 (B) hemocytes (arrows; +LTP; right blot panel).