Microvesicles and membranes display discrete glycomes
We hybridized 1.5 μg (based on protein concentration) of either Cy3-labeled microvesicles or cell membrane preparations to our lectin microarrays. Electron microscopy images of general cell membrane preparations from Sk-Mel-5 revealed a size distribution of vesicle-like structures (20-200 nm) with a few larger membranous structures (). The majority of particles observed were under 100 nm in size, comparable to our microvesicle samples. Both enrichment and exclusion of glycan epitopes were observed in microvesicles when compared to their parent membrane preparations as measured by fluorescence intensities (). In general, lectin analysis revealed that microvesicles were enriched in high mannose (HHL, PSA, NPA, SVN, UDA), complex N
-linked glycan (Calsepa,32
LcH, PHA-E, PHA-L, TL), α-2,6 sialic acid (SNA, TJA-I) and poly N
-acetyllactosamine (LEA, DSA) epitopes but showed little to no binding to lectins that recognize terminal N
-acetyl-D-galactosamine (CAA, DBA, SBA, SJA, BPA) and Blood Group A/B (EEA, LTL). An example of the raw data is shown in and Supplementary Figure 2
for the SkMel-5 cell line.
Figure 2 SkMel-5 microvesicles and cell-membranes have discrete glycomic profiles. (A) Equal amounts (1.5 μg by protein) of Cy3-labeled samples were added to the lectin microarray. Lectins were hierarchically clustered by using the Pearson correlation (more ...)
Single color analysis can only distinguish differences in carbohydrate levels between samples if lectin binding is in the linear range.17, 24
In SkMel-5, WGA, TJA-I (sialic acid binding lectins), NPA (a high mannose lectin), and DSA (a polyLacNAc binder) appear to bind microvesicles and the corresponding cell membrane preparations at similar levels as measured by the median fluorescence intensity, which is irrespective of spot size and morphology (). However, lectins with similar specificity profiles (α-2,6 sialic acid: SNA ; high mannose: HHL, PSA) show clear enrichment in binding to the microvesicles, suggesting that some lectins are not in the linear range of detection. To address this issue and validate our single color results, we utilized the ratiometric dual-color approach developed in our laboratory.17, 24
In this approach, competitive binding between the sample and an orthogonally labeled common reference enables comparison of the relative binding levels thereby increasing sensitivity to differences between samples. We used Cy5-labeled H9 cell membrane preparations as a biological reference for our comparative analysis. Glycoprofiles of our panel of microvesicles and parent cell membranes were obtained by incubation of 1.5 μg of Cy3-labeled sample against an equal amount of reference (). Using this method, we observed the expected enrichment of DSA, WGA, NPA and TJA-I epitopes in SkMel-5 microvesicles ().
Figure 3 Ratiometric lectin microarray comparison using H9 membrane as a reference. (A) Experimental schematic. Equal amounts of Cy3-labeled MV and parent MB were incubated against Cy5-labeled H9 MB. (B) Median-normalized log2 ratios (Sample/Reference) of the (more ...)
Hierarchical clustering of the arrays using the Pearson correlation coefficient as the distance metric and average linkage analysis revealed a conserved lectin binding pattern for our panel of microvesicles, including those derived from the physiological fluid, breast milk (, R=0.57, n
=75, P < 0.0001, two-tailed
). In contrast, clustering of the parent cell membranes did not show a conserved glycomic signature (R = 0.25, n
= 75, P = 0.03). The data shown is median-centered to aid in the comparison between differentially labeled samples.29
Uncentered data gives similar correlation coefficients (Supplementary Figures 3
). In this work we consider a P value less than or equal to 0.0001 as our threshold for significance to indicate a conserved glycome. Our data is consistent with our previous work on T-cell microvesicles (Jurkat, H9 and SupT1) and suggests that the glycomic pattern observed for microvesicles is not a phenomenon of the culture conditions.17
Our analysis revealed two sets of GalNAc-specific lectins with divergent behaviors. The first set (DBA, BPA, SBA, VVA), identified in our single color analysis, bind to terminal α- and β-GalNAc epitopes, such as Blood Group A. They display lower binding to the microvesicles, arguing that the epitopes they bind to are excluded. Heirarchichal clustering of the lectins in our dual-color analysis identified a second cohort of GalNAc binders (MNA-G, MPA, SNA-II, IRA, HPA, AIA) specific for terminal α-GalNAc structures such as the Tn antigen, with highly enriched binding to the microvesicles. Overall, the microvesicle signature observed was consistent with our single color data, revealing a glycomic pattern of enrichment in high mannose, complex N-linked glycans and polyLacNAc epitopes and a reduction in Blood Group A/B antigens. The enriched epitopes have been associated with apical trafficking of glycoproteins by endogenous lectins including galectins, suggesting that glycans may be involved in trafficking to these particles.
Microvesicles displayed some cell specific differences in glycosylation. For example, neither SkMel-5 microvesicles nor the parent cell membranes show binding to Blood Group H binding lectins (UEA-I, PTL-II, TJA-II). However this epitope, which is present in HCT-15 membranes, is clearly enriched in HCT-15 microvesicles. In addition, HCT-15 and HT29 cells, which have diminished levels of α-2,6 sialic acid in their membranes when compared to the entire panel of cell lines, consequently displayed lower levels overall of this epitope in their cognate microvesicles (SNA, SNA-I and TJA-I, ), although enrichment is still observed in comparison to the parent membranes. Thus, the gross glycomic composition of the parent membrane is reflected in the microvesicle glycome.
We performed multiple control experiments to confirm the specificity of lectin binding to our microvesicle samples. First, we inhibited binding on our arrays by pre-incubation of the lectins with the appropriate mono- and disaccharides and observed the expected results overall (see Supplementary Figure 5
). It should be noted that some lectins, especially complex epitope binders such as TL, are not inhibited by simple sugars. Next we subjected our microvesicles to enzymatic treatments to gain further insight into the specificity of lectin binding. Treatment of SkMel-5 microvesicles with Endo Hf
, an enzyme that cleaves within the core of high mannose and hybrid N
-linked glycans, preferentially inhibited binding to mannose lectins (NPA, HHL, ). In contrast, the enzyme PNGase F, which cleaves all mammalian N
-linked glycans, significantly reduced the binding of lectins to both mannose and complex epitopes as expected (). A strong reduction was also observed in α-2,6 sialic acid binding (SNA, TJA-I) upon PNGaseF treatment, arguing that this epitope is mainly in N
-linked glycans on the microvesicles. The specificity of these lectins (SNA, TJA-I) for sialic acid was confirmed by the loss of microvesicle binding to these lectins upon treatment with neuraminidase (sialidase), which cleaves this glycan (Supplementary Figure 6
). Disparate results were seen upon treatment with PNGaseF for the polyLacNAc binders DSA and LEA. While a large reduction in binding upon N
-glycan cleavage was observed for DSA (78%), LEA displayed only a modest reduction (17%). Although both bind to polyLacNAc, the two lectins have discrete specificities, with LEA preferring long polyLacNAc chains.33, 34
Our data indicates that polyLacNAc may be present in multiple contexts within SkMel-5 microvesicles, including possible O
-linked and glycolipid epitopes.
Figure 4 Enzymatic treatment of microvesicles confirms specificity of lectins. Cy3-labeled SkMel-5 MV were treated with (A) EndoHf and (B) PNGaseF for 18 h prior to hybridization on the array. Fluorescence values are expressed as a mean ± SD of replicate (more ...)
Microvesicle enrichment in conserved glycans suggests carbohydrate-mediated sorting
To directly examine whether our observed microvesicle signature could result from glycan-mediated sorting to microvesicles, we compared microvesicles and their cognate membranes using our dual-color assay. Equal amounts of Cy3-labeled microvesicles and their corresponding Cy5-labeled parent cell membranes were combined and incubated on our lectin microarrays. We again observed the previous pattern of enrichment and exclusion of specific glycans as a dominant feature of the microvesicle glycosylation profiles when compared to their parent membranes (). This implies that the glycan signature is due to sorting of glycoproteins and glycolipids to the microvesicles, though whether this is due to the glycans themselves is not known. Although there was no common biological reference, heirarchical clustering using the Pearson correlation and average linkage analysis again showed a clear glycopattern (, R = 0.66, n
= 72, P < 0.0001 and Supplementary Figure 7
). We validated the enrichment and exclusion of epitopes by lectin blot analysis (Supplementary Figures 8
). This data strongly suggests that microvesicles are emerging from a specific portion of the membrane with a conserved glycome.
Figure 5 Direct comparison of microvesicles with their parent cell-membranes. (A) Experimental schematic. Equal amounts of Cy3-labeled MV were analyzed against Cy5-labeled parent MB on the lectin microarray. (B) Median-centered log2 ratios were hierarchically (more ...)
The microvesicle glycome could arise from the presence of predominant conserved glycoproteins or lipids containing the same glycoforms. These could be concentrated in microvesicles by mechanisms other than glycan-mediated sorting. To examine this possibility, we performed a lectin blot assay with DSA, a lectin whose binding epitope, polyLacNAc, is enriched in microvesicles (). Equal amounts (by protein) of microvesicles and membranes from 3 cell lines (SkMel-5, HT-29 and Jurkat) were probed. Enrichment in DSA binding was observed in the microvesicles in concordance with the microarray data (, and Supplementary Figures 9
). However, the microvesicles from each cell line showed a distinct pattern of DSA-positive bands. This provides preliminary evidence that different glycoproteins are responsible for DSA-reactivity within the microvesicles from distinct cell lines.
Figure 6 A possible role for glycans in MV protein sorting. (A) DSA lectin blot of equal protein amounts of SkMel-5, Jurkat and HCT-15 MV and MB shows enrichment of DSA epitopes on multiple proteins in MV. (B) The cell derived MV were probed for galectin-3 and (more ...)
Multiple lectins have been implicated in carbohydrate-based protein sorting to specific membranes. Among these lectins, galectins -3 and -4, which bind LacNAc epitopes, have been identified in microvesicles.35, 36
These galectins are known to mediate glycoprotein sorting to apical domains in polarized epithelia cells.37, 38
We tested for the presence of galectins -3 and -4 by Western blot analysis (). Galectin-3 was observed in microvesicles derived from two of our cell lines, SkMel-5 and HCT-15. We found galectin-4 in the microvesicles of the T-cell line H9 and the colon cancer cell line HT29. Microvesicles derived from the other two T-cell lines, Jurkat and SupT1, did not display either of these galectins, although other galectin isoforms may be present. We do not yet know whether these lectins are playing a role in the origination of our observed microvesicle glycan signature. However, their presence in four of our six microvesicle samples, coupled with our observation that polyLacNAc epitopes are enriched in these particles suggests that these lectins may be involved, a subject of future research.