Unique, Polyfucosylated Glycan–Receptor Interactions Are Essential for Regeneration of <i>Hydra magnipapillata</i>

Cell–cell communications, cell–matrix interactions, and cell migrations play a major role in regeneration. However, little is known about the molecular players involved in these critical events, especially cell surface molecules. Here, we demonstrate the role of specific glycan–receptor interactions in the regenerative process using <i>Hydra magnipapillata</i> as a model system. Global characterization of the <i>N</i>- and <i>O</i>-glycans expressed by <i>H. magnipapillata</i> using ultrasensitive mass spectrometry revealed mainly polyfucosylated LacdiNAc antennary structures. Affinity purification showed that a putative C-type lectin (accession number Q6SIX6) is a likely endogenous receptor for the novel polyfucosylated glycans. Disruption of glycan–receptor interactions led to complete shutdown of the regeneration machinery in live <i>Hydra</i>. A time-dependent, lack-of-regeneration phenotype observed upon incubation with exogenous fuco-lectins suggests the involvement of a polyfucose receptor-mediated signaling mechanism during regeneration. Thus, for the first time, the results presented here provide direct evidence for the role of polyfucosylated glycan–receptor interactions in the regeneration of <i>H. magnipapillata.</i

Effect of the <i>S</i>. <i>nigra</i> RIPs and lectins on protein synthesis in a cell-free translation assay.

<p>(A) Dose response curves of luciferase synthesis in treatments with SNA-I (non-reduced and reduced) and lectins (SNA-II, SNA-IV). (B and C) Dose response curves of luciferase synthesis in the treatments with non-reduced and reduced RIP for SNA-V and SNLRP, respectively. (D) IC50 values for the RIPs and lectins.</p

Confocal microscopic images and quantitative analysis of the colocalization.

<p>(A) Double immunofluorescence analysis of FITC-labeled SNA-I (a, e, i and m), and marker for ER (b, c and d), Golgi (f, g and h), endosomes (j, k and l) and lysosomes (n, o and p) in HeLa cells. The merged reconstructed images are shown in (d, h, l and p) with the green dots from FITC-labeled SNA-I and the red dots from the marker. The arrow indicates SNA-I dots overlapping with the marker. Scale bars represent 10 μm. (B) Manders’ coefficients and object-based colocalization graphs of the colocalization image analysis study. Asterisks denote values significantly different from the lysosome (*: p < 0,05; **: p< 0,01; ***: p < 0,001).</p

Structures of glycans derived from glycolipids observed in the MALDI-TOF MS spectra of Hela and NHDF cells.

<p>All glycans are deuteroreduced (DR), permethylated and shown in the form of [M+Na]⁺. These glycan structures and linkages are drawn based on the molecular weight, glycolipid glycan biosynthetic pathway and MS/MS data. ND, not detected. * = minor (<20%), ** = medium (20–50%), *** = major (>50%).</p

Internalization of FITC labeled <i>S</i>. <i>nigra</i> proteins in HeLa cells.

<p>(A) Confocal microscopic images of the uptake of SNA-I (25 nM) in HeLa cells after 6 hours incubation. The arrow indicates a spot where protein is accumulating. Nuclei have been delineated be in white. Scale bars represent 10 μm. (B) Uptake of 50 nM FITC labeled SNA-I, SNA-II, SNA-IV, SNA-V and SNLRP by HeLa cells after 0 min, 5 min, 30 min, 1 h, 3 h, 6 h, 9 h and 12 h incubation, based on fluorescence intensity, which was normalized by the FITC labeling efficiency. The pictures for quantification were acquired using identical confocal settings. Data are given as mean ± SE, based on at least 80 individual cell measurements per sample and each treatment was carried out with three independent replicates.</p

Validation of GALK1 and GALE+GALK1 deficient knockout cell lines.

<p>Following single cell sort, potential knockout clones were allowed to grow until they reached confluence in 6 well plates. GALK1 enzymatic activity was analyzed on the protein level. HEK293T, GALE KO, GALK1 KO cells (clone 10), and GALE+GALK1 double KO cells (clone 12) were transfected with expression vectors for SIVmac239 gp120 made as a truncated secreted product with a C-terminal polyhistidine tag. Secreted gp120 was purified from supernatant 48 hours post transfection using nickel-NTA columns. 3μg of purified gp120 was run on two 4–12% SDS-PAGE gels in duplicate. The first gel was probed for gp120 using the rhesus anti-gp120 monoclonal antibody 3.11H. The second gel was probed for O-glycosylation using an HRP labeled Jacalin lectin. For protein production, cells were grown in serum free media, serum free media +galactose, or serum free media +galactose +GalNAc as indicated. For further detail on cell culture conditions, refer to the materials and methods.</p

Comparison of LC50 values for the <i>S</i>. <i>nigra</i> proteins in HeLa and NHDF cell lines.

<p>Data are shown as means ± SE based on 4 replications per treatment, and each experiment was repeated 3 times. Different letters (a-h) represent significant cytotoxicity differences (Duncan; <i>P</i> < 0.05) between different <i>S</i>. <i>nigra</i> proteins under each treatment.</p><p>Comparison of LC50 values for the <i>S</i>. <i>nigra</i> proteins in HeLa and NHDF cell lines.</p

Dose response curve of the effect of <i>S</i>. <i>nigra</i> proteins on HeLa and NHDF cell viability after 24 and 48 h.

<p>(A) Dose-response curves for HeLa cells incubated for 24 and 48h with different concentrations of <i>S</i>. <i>nigra</i> RIPs/lectins. (B) Log concentration – cell viability curve of NHDF cells incubated for 24 and 48 h with different concentrations of <i>S</i>. <i>nigra</i> RIPs/lectins. % ctrl (treated/control X 100) = ratio of surviving treated cells/ surviving cells percent in control. All data are expressed as means ± SE of 3 biological replicates in 4 technical replicates (n = 12). (C) Transmission light microscopy images of HeLa cells grown in the absence (control) and presence of 1.5 μM SNA-V for 24 h. Scale bars represent 100 μm.</p

<i>In cellula</i> protein translation inhibition activity of <i>S</i>. <i>nigra</i> RIPs (A) Merged confocal images of HeLa cells stained with DAPI (blue) and BODIPY (green).

<p>Scale bars represent 15 μm. (B) Average numbers of LDs/cell (n>400) (C) Luciferase activity measured in HG1-luc2-IRES-tCD cells incubated with <i>S</i>. <i>nigra</i> RIPs/lectins and controls. The treatment with 1 x PBS was selected as the negative control, and cycloheximide was used as a positive control. The luminescent signal was normalized by the fluorescence signal from the Presto blue assay to correct for variations in cell density. Asterisks denote values significantly different from the cells incubated with control (1 x PBS) (*: p < 0,05; **: p< 0,01; ***: p < 0,001).</p

Validation of GALK2 and GALE+GALK2 deficient knockout cell lines.

<p>Following single cell sort, potential knockout clones were allowed to grow until they reached confluence in 6 well plates. <b>A)</b> Cell lysate was harvested by incubating cells in RIPA buffer. Clarified lysate was loaded onto a 4–12% SDS-PAGE gel and probed with an anti-GALK2 antibody. Parental GALE KO cell lysate was loaded in the first lane as a control. <b>B)</b> GALK2 enzymatic activity was analyzed at the protein level. GALK2 KO cells (clone 7) and GALE+GALK2 double KO cells (clone 7) were transfected with expression vectors for SIVmac239 gp120 made as a truncated secreted product with a C-terminal polyhistidine tag. Secreted gp120 was purified from supernatant 48 hours post transfection using nickel-NTA columns. 3μg of purified gp120 was run on three 4–12% SDS-PAGE gels in triplicate. The first was analyzed by Coomassie Blue staining. The second gel was probed for gp120 using the rhesus anti-gp120 monoclonal antibody 3.11H. The third gel was probed for O-glycosylation using an HRP labeled Jacalin lectin. For protein production, cells were grown in serum free media, serum free media +galactose +GalNAc (+Sugars), or serum free media +UDP-galactose +UDP-GalNAc (+UDP) as indicated. For further detail on cell culture conditions, refer to the materials and methods.</p