Extensive determination of glycan heterogeneity reveals an unusual abundance of high mannose glycans in enriched plasma membranes of human embryonic stem cells.

Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine cell surface glycosylation.

Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells.

Most clinically approved biomarkers of cancer are glycoproteins, and those residing on the cell surface are of particular interest in biotherapeutics. We report a method for selective labeling, affinity enrichment, and identification of cell-surface glycoproteins. PC-3 cells and primary human prostate cancer tissue were treated with peracetylated N-azidoacetylgalactosamine, resulting in metabolic labeling of cell surface glycans with the azidosugar. We used mass spectrometry to identify over 70 cell surface glycoproteins and biochemically validated CD146 and integrin beta-4, both of which are known to promote metastatic behavior. These results establish cell-surface glycoproteomics as an effective technique for discovery of cancer biomarkers.

Effects of heterocyclic aromatic substituents on binding affinities at two distinct sites of somatostatin receptors. Correlation with the electrostatic potential of the substituents.

In our continuing program exploring glucose-based peptidomimetics of somatostatin (SRIF-14), we sought to improve the water solubility of our glycosides. This led to insights into the nature of the ligand binding sites at the SRIF receptor. Replacement of the C4 benzyl substituent in glucoside (+)-2 with pyridinylmethyl or pyrazin-2-ylmethyl congeners increased water solubility and enhanced affinity for the human SRIF subtype receptor 4 (sst4). We attribute this effect to hydrogen bond formation. The pyridin-3-ylmethyl substituent at C4, when combined with the imidazol-4-ylmethyl group at C2, generated (-)-19, which has the highest affinity of a glucose-based peptidomimetic at a human SRIF receptor to date (K(i) 53 +/- 23 nM, n = 6 at sst4). The C4 heterocyclic congeners of glucosides bearing a 1-methoxy substituent rather than an indole side chain at the anomeric carbon, such as (+)-16, also provided information about the Trp(8) binding pocket. We correlated the SARs at both the C4 and the Trp(8) binding pockets with calculations of the electrostatic potentials of the diverse C4 aromatic substituents using Spartan 3-21G(*) MO analysis. These calculations provide an approximate analysis of a molecule's ability to interact within a receptor binding site. Our binding studies show that benzene and indole rings, but not pyridinylmethyl nor pyrazin-2-ylmethyl rings, can bind the hydrophobic Trp(8) binding pocket of sst4. The Spartan 3-21G(*) MO analysis reveals significant negative electrostatic potential in the region of the pi-clouds for the benzene and indole rings but not for the pyridinylmethyl or pyrazin-2-ylmethyl congeners. Our data further demonstrate that the replacement of benzene or indole side chains by heterocyclic aromatic rings typified by pyridine and pyrazine not only enhances water solubility and hydrogen bonding capacity as expected, but can also profoundly diminish the ability of the pi-cloud of the aromatic substituent to interact with side chains of an aromatic binding pocket such as that for Trp(8) of SRIF-14. Conversely, these calculations accommodate the experimental findings that pyrazin-2-ylmethyl and pyridinylmethyl substituents at C4- of C1-indole-substituted glycosides afford higher affinities at sst4 than the C4-benzyl group of (+)-2. This result is consistent with the high electron density in the plane of the heterocycle depicted in Figure 6 which can accept hydrogen bonds from the C4 binding pocket of the receptor. Unexpectedly, we found that the 2-fluoropyridin-5-ylmethyl analogue (+)-14 more closely resembles the binding affinity of (+)-8 than that of (+)-2, thus suggesting that (+)-14 represents a rare example of a carbon linked fluorine atom acting as a hydrogen bond acceptor. We attribute this result to the ability of the proton to bind the nitrogen and fluorine atoms simultaneously in a bifurcated arrangement. At the NK1 receptor of substance P (SP), the free hydroxyl at C4 optimizes affinity.

Mucin granule intraluminal organization in living mucous/goblet cells. Roles of protein post-translational modifications and secretion.

Recent studies suggest that the mucin granule lumen consists of a matrix meshwork embedded in a fluid phase. Secretory products can both diffuse, although very slowly, through the meshwork pores and interact noncovalently with the matrix. Using a green fluorescent protein-mucin fusion protein (SHGFP-MUC5AC/CK) as a FRAP (fluorescence recovery after photobleaching) probe, we have assessed in living mucous cells the relative importance of different protein post-translational modifications on the intragranular organization. Long term inhibition of mucin-type O-glycosylation, sialylation, or sulfation altered SHGFP-MUC5AC/CK characteristic diffusion time (t(1/2)), whereas all but sulfation diminished its mobile fraction. Reduction of protein disulfide bonds with tris(hydroxypropyl)phosphine resulted in virtually complete immobilization of the SHGFP-MUC5AC/CK intragranular pool. However, when activity of the vacuolar H+-ATPase was also inhibited, disulfide reduction decreased SHGFP-MUC5AC/CK t((1/2)) while diminishing its intraluminal concentration. Similar FRAP profiles were observed in granules that remained in the cells after the addition of a mucin secretagogue. Taken together these results suggest that: (a) the relative content of O-glycans and intragranular anionic groups is crucial for protein diffusion through the intragranular meshwork; (b) protein-protein, rather than carbohydrate-mediated, interactions are responsible for binding of SHGFP-MUC5AC/CK to the immobile fraction, although the degree of matrix O-glycosylation and sialylation affects such interactions; (c) intragranular organization does not depend on covalent multimerization of mucins or the presence of native disulfide bonds in the intragranular mucin/proteins, but rather on specific protein-mediated interactions that are important during the early stages of mucin matrix condensation; (d) alterations of the intragranular matrix precede granule discharge, which can be partial and, accordingly, does not necessarily involve the disappearance of the granule.