A role for the L-selectin adhesion system in mediating cytotrophoblast emigration from the placenta

Cytotrophoblast (CTB) aggregates that bridge the gap between the placenta and the uterus are suspended as cell columns in the intervillous space, where they experience significant amounts of shear stress generated by maternal blood flow. The proper formation of these structures is crucial to pregnancy outcome as they play a vital role in anchoring the embryo/fetus to the decidua. At the same time, they provide a route by which CTBs enter the uterine wall. The mechanism by which the integrity of the columns is maintained while allowing cell movement is unknown. Here, we present evidence that the interactions of L-selectin with its carbohydrate ligands, a specialized adhesion system that is activated by shear stress, play an important role. CTBs in cell columns, particularly near the distal ends, stained brightly for L-selectin and with the TRA-1-81 antibody, which recognizes carbohydrate epitopes that support binding of L-selectin chimeras in vitro. Function-perturbing antibodies that inhibited either receptor or ligand activity also inhibited formation of cell columns in vitro. Together, these results suggest an autocrine role for the CTB L-selectin adhesion system in forming and maintaining cell columns during the early stages of placental development, when the architecture of the basal plate region is established. This type of adhesion may also facilitate CTB exit from cell columns, a prerequisite for uterine invasion.     

Lectin chromatography/mass spectrometry discovery workflow identifies putative biomarkers of aggressive breast cancers

We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n=5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, ～90% of which were components of the cell surface or extracellular matrix. We observed 1011 glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNaseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were up-regulated in triple negative vs luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype.     

Glycosylation of human trophoblast integrins is stage and cell-type specific

Cytotrophoblasts are the specialized epithelial cells of theplacenta. During the first trimester, a subpopulation of chorionicvillas cytotrophoblasts differentiates along an invasive pathwayand penetrates the maternal endo-metrium, decidua and spiralarterioles. Cytotrophoblast invasiveness declines rapidly duringthe second half of gestation. Isolated cytotrophoblasts of differentgestational ages retain this differential invasiveness in culture.To determine whether the properties of integrin receptors forextracellular matrix molecules differ between invasive and non-invasivecytotrophoblasts, detergent extracts of isolated cytotrophoblastsof different gestational ages, and of first-trimester villousfibroblasts, were immunoprecipitated with subunit-specific anti-     

Biochemical and biophysical comparison of two mucins from human submandibular-sublingual saliva

A high-molecular-weight mucin-glycoprotein (MG1) was isolated from human submandibular-sublingual saliva and was comprised of 14.9% protein, 29.0% N-acetylglucosamine, 9.4% N-acetylgalactosamine, 10.5% fucose, 24.2% galactose, 0.9% mannose, 4.0% N-acetylneuraminic acid, and 7.0% sulfate. Carbohydrate units were O-glycosidically linked and ranged in size from 4 to 16 residues. The biophysical properties of MG1 were compared to those of a smaller mucin (MG2) also isolated from submandibular-sublingual saliva. Fluorescence spectroscopy demonstrated that MG1 bound both 1-anilino-8-naphthalenesulfonate (ANS) and N-phenyl-1-naphthylamine (NPNA) in stable hydrophobic binding sites (melting temperature, 47 +/- 2 degrees C), whereas MG2 did not bind these hydrophobic probes. These hydrophobic domains occurred on nonglycosylated or naked portions of MG1 since Pronase treatment eliminated ANS binding. Reduction of disulfide bridges in MG1 increased the number of available hydrophobic binding sites. High ionic strength (0 to 2 M NaCl) had no effect on ligand binding, whereas lowering pH (9 to 2) increased ANS binding without affecting NPNA complexation. Circular dichroism (CD) data suggested that MG1's carbohydrate chains dominated its spectrum. In contrast, the peptide backbone dominated the CD spectrum of MG2. Collectively, the results of this study indicate that human submandibular-sublingual saliva contains two structurally distinct mucins.     

Toward defining the human parotid gland salivary proteome and peptidome: identification and characterization using 2D SDS-PAGE, ultrafiltration, HPLC, and mass spectrometry

Saliva plays many biological roles, from lubrication and digestion to regulating bacterial and leukocyte adhesion. To understand the functions of individual components and families of molecules, it is important to identify as many salivary proteins as possible. Toward this goal, we used a proteomic approach as the first step in a global analysis of this important body fluid. We collected parotid saliva as the ductal secretion from three human donors and separated the protein components by two-dimensional SDS-polyacrylamide gel electrophoresis (2D SDS-PAGE). Proteins in gel spots were identified by peptide mass fingerprinting, and the results were confirmed by tandem mass spectrometry of selected peptides. Complementing this approach we used ultrafiltration to prepare a low-molecular-weight fraction of parotid saliva, which was analyzed directly or after reversed phase high-performance liquid chromatography separation by using mass spectrometric approaches. MS analyses of 2D SDS-PAGE spots revealed known components of saliva, including cystatins, histatins, lysozyme, and isoforms and/or fragments of alpha-amylase, albumin, and proline-rich proteins. We also discovered novel proteins, such as several isoforms of Zn-alpha-2-glycoprotein and secretory actin-binding protein. MS analyses of the ultrafiltrate showed that the low-molecular-weight fraction of parotid saliva was peptide-rich, with novel fragments of proline-rich proteins and histatins in abundance. Experiments using Candida albicans as the test organism showed that at least one of the novel peptides had antifungal activity. Our results show that saliva is a rich source of proteins and peptides that are potential diagnostic and therapeutic targets.     

The proteomes of human parotid and submandibular/sublingual gland salivas collected as the ductal secretions

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.     

Bacterial adherence on replicas of sodium dodecyl sulfate-polyacrylamide gels

A method for determining which molecules in a complex mixture of proteins can function as bacterial receptors was devised. Salivary proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Bacteria that were metabolically labeled with 3H or externally labeled with 125I were incubated on the nitrocellulose replicas. After 18 h at 4 degrees C, the unbound cells were removed by repeated washing of the replicas, and the bands to which the radiolabeled bacteria bound were visualized by autoradiography. By this technique, Fusobacterium nucleatum, which adheres via carbohydrate residues on receptor molecules, and Staphylococcus aureus, which recognizes the peptide portion of fibronectin, were shown to bind specifically to their respective receptors. These results suggest that this method can be useful for profiling bacterial binding to either the carbohydrate or the protein portions of molecules present in complex mixtures, such as those composing biological fluids or tissue substrates. Structural specificities, such as recognition sequences formed by certain oligosaccharides, could be further investigated by adding the appropriate simple sugars, as well as oligosaccharide inhibitors, to the incubation medium. The latter approach is particularly important since most glycoproteins carry multiple N- and O-linked carbohydrate substituents that could serve as bacterial receptors.     

Structure and bacterial receptor activity of a human salivary proline-rich glycoprotein

Using an overlay technique, we previously showed that the Gram-negative periodontal pathogen Fusobacterium nucleatum binds to a glycoprotein of Mr 89,000 (Prakobphol, A., Murray, P., and Fischer, S.J. (1987) Anal. Biochem. 164, 5-11) in the parotid saliva of some individuals. We now show that deglycosylation of the purified glycoprotein results in loss of receptor activity. Amino acid analysis of the protein core showed predominantly proline, glycine, and glutamic acid/glutamine, a characteristic of proline-rich glycoproteins (PRG). The amino terminus contained repeating sequences of Ser-Gln-Gly-Pro-Pro-Pro-Arg-Pro-Gly-Lys-Pro-Glu-Gly-Pro-Pro-Pro- Gln-Gly that had significant compositional and sequence homology to that encoded by exon 3 of the PRB3 gene. We analyzed the PRG oligosaccharides by a combination of mass spectrometry techniques and nuclear magnetic resonance spectroscopy. Twenty-seven highly fucosylated structures were identified. The most abundant was as follows (where Fuc is fucose). (formula; see text) To understand the structural basis of F. nucleatum binding, we screened glycolipids and neoglycolipids carrying carbohydrate structures related to those of the PRG for receptor activity; components with unsubstituted terminal lactosamine residues best supported adherence. Neoglycolipids constructed from PRG oligosaccharides were also receptors. Treatment with beta-galactosidase, but not alpha-fucosidase, abolished binding, suggesting that unsubstituted lactosamine units, including the 6-antenna of the major oligosaccharide, mediate F. nucleatum adherence.     

Highly glycosylated human salivary molecules present oligosaccharides that mediate adhesion of leukocytes and Helicobacter pylori

Glycoproteins display carbohydrate facets that serve as adhesion receptors for cells including leukocytes and bacterial cells. Our aim was to understand the role of the specialized carbohydrate motifs carried by highly glycosylated human salivary proteins in regulating the oral ecology. To date, our structural studies suggest that these molecules display a wide array of oligosaccharide structures, including many species with highly charged and/or fucosylated termini. Here, we used an immunoblot approach to gain additional information about the nature of these oligosaccharides. The results showed that MG1 and the salivary agglutinin express the MECA-79 epitope, an unusual sulfated carbohydrate structure that belongs to an important class of high-affinity (endothelial) L-selectin ligands. Unexpectedly, we discovered that in many women the expression of this epitope is hormonally regulated. Additional experiments revealed that MG1, MG2, and the salivary agglutinin also present Lewis blood group antigens, the exact repertoire varying on an individual basis. In parallel, we explored the functions of these carbohydrate motifs. Using an assay that detects L-selectin ligands, we found that the subset of MECA-79-reactive oligosaccharides displayed on salivary molecules specifically bind an L-selectin/Fc chimera. In contrast, the Lewis blood group structures are receptors for many strains of Helicobacter pylori, an organism that is implicated in the development of gastric ulcers and cancer. Together, these results suggest that MG1, MG2, and the salivary agglutinin play important roles in governing leukocyte and bacterial adhesion. Our findings suggest novel strategies, based on the relevant carbohydrate structures, for promoting or inhibiting these processes.     

The salivary mucin MG1 (MUC5B) carries a repertoire of unique oligosaccharides that is large and diverse

The high-molecular-mass salivary mucin MG1, one of two major mucins produced by human salivary glands, plays an important role in oral health by coating the tooth surface and by acting as a bacterial receptor. Here this mucin was purified from the submandibular/sublingual saliva of a blood group O individual. The presence of MUC5B as the major mucin in this preparation was confirmed by amino acid analysis and its reactivity with the monoclonal antibody PAN H2. To structurally characterize MG1 carbohydrates the O-glycans were released by reductive beta-elimination. Nuclear magnetic resonance spectroscopy of the nonfractionated mixture showed that (1) fucose was present in blood group H, Le(a), Le(x), Le(b), and Le(y) epitopes; (2) NeuAc was mainly linked alpha 2-3 to Gal or alpha 2-6 to GalNAcol; and (3) the major internal structures were core 1 and core 2 sequences. After this preliminary analysis the released oligosaccharides were separated into neutral (56%), sialylated (26%), and sulfated (19%) fractions, with an average length of 13, 17, and 41 sugar residues, respectively. Gas chromatography-mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of mixtures of neutral and sialylated oligosaccharides revealed at least 62 neutral and 25 sialylated oligosaccharides consisting of up to 20 monosaccharide residues. These results showed that the MG1-derived oligosaccharides were much longer than those of MG2, and only a few species were found on both molecules. Thus, these two mucins create an enormous repertoire of potential binding sites for microorganisms at one of the major portals where infectious organisms enter the body.