Integrin α4-positive human trophoblast progenitors: functional characterization and transcriptional regulation.

STUDY QUESTION: What are the functional characteristics and transcriptional regulators of human trophoblast progenitor cells (TBPCs)? SUMMARY ANSWER: TBPC lines established from the human smooth chorion by cell sorting for integrin α4 expressed markers of stemness and trophoblast (TB) stage-specific antigens, invaded Matrigel substrates and contributed to the cytotrophoblasts (CTBs) layer of smooth chorion explants with high-mobility group protein HMGI-C (HMGA2) and transcription factor GATA-4 (GATA4) controlling their progenitor state and TB identity. WHAT IS KNOWN ALREADY: Previously, we reported the derivation of TBPC lines by trypsinization of colonies that formed in cultures of chorionic mesenchyme cells that were treated with an activin nodal inhibitor. Microarray analyses showed that, among integrins, α4 was most highly expressed, and identified HMGA2 and GATA4 as potential transcriptional regulators. STUDY DESIGN, SIZE, DURATION: The aim of this study was to streamline TBPC derivation across gestation. High-cell surface expression of integrin α4 enabled the use of a fluorescence-activated cell sorter (FACS) approach for TBPC isolation from the human smooth chorion (n = 6 lines). To confirm their TBPC identity, we profiled their expression of stemness and TB markers, and growth factor receptors. At a functional level, we assayed their invasive capacity (n = 3) and tropism for the CTB layer of the smooth chorion (n = 3). At a molecular level, we studied the roles of HMGA2 and GATA4. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Cells were enzymatically disassociated from the human smooth chorion across gestation. FACS was used to isolate the integrin α4-positive population. In total, we established six TBPC lines, two per trimester. Their identity was determined by immunolocalization of a suite of antigens. Function was assessed via Matrigel invasion and co-culture with explants of the human smooth chorion. An siRNA approach was used to down-regulate HMGA2 and GATA4 expression and the results were confirmed by immunoblotting and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses. The endpoints analyzed included proliferation, as determined by 5-bromo-2'-deoxyuridine (BrDU) incorporation, and the expression of stage-specific antigens and hormones, as determined by qRT-PCR and immunostaining approaches. MAIN RESULTS AND THE ROLE OF CHANCE: As with the original cell lines, the progenitors expressed a combination of human embryonic stem cell and TB markers. Upon differentiation, they primarily formed CTBs, which were capable of Matrigel invasion. Co-culture of the cells with smooth chorion explants enabled their migration through the mesenchyme after which they intercalated within the chorionic CTB layer. Down-regulation of HMGA2 showed that this DNA-binding protein governed their self-renewal. Both HMGA2 and GATA4 had pleitropic effects on the cells' progenitor state and TB identity. LIMITATIONS, REASONS FOR CAUTION: This study supported our hypothesis that TBPCs from the chorionic mesenchyme can contribute to the subpopulation of CTBs that reside in the smooth chorion. In the absence of in vivo data, which is difficult to obtain in humans, the results have the limitations common to all in vitro studies. WIDER IMPLICATIONS OF THE FINDINGS: The accepted view is that progenitors reside among the villous CTB subpopulation. Here, we show that TBPCs also reside in the mesenchymal layer of the smooth chorion throughout gestation. We theorize that they can contribute to the CTB layer in this region. This phenomenon may be particularly important in pathological situations when CTBs of the smooth chorion might provide a functional reserve for CTBs of the placenta proper. STUDY FUNDING/COMPETING INTERESTS: Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under award P50HD055764. O.G., N.L., K.O., A.P., T.G.-G., M.K., A.B., M.G. have nothing to disclose. S.J.F. received licensing fees and royalties from SeraCare Life Sciences for trisomic TBPC lines that were derived according to the methods described in this manuscript. TRIAL REGISTRATION NUMBER: N/A.

Salivary agglutinin, which binds Streptococcus mutans and Helicobacter pylori, is the lung scavenger receptor cysteine-rich protein gp-340.

Salivary agglutinin is a high molecular mass component of human saliva that binds Streptococcus mutans, an oral bacterium implicated in dental caries. To study its protein sequence, we isolated the agglutinin from human parotid saliva. After trypsin digestion, a portion was analyzed by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), which gave the molecular mass of 14 unique peptides. The remainder of the digest was subjected to high performance liquid chromatography, and the separated peptides were analyzed by MALDI-TOF/post-source decay; the spectra gave the sequences of five peptides. The molecular mass and peptide sequence information showed that salivary agglutinin peptides were identical to sequences in lung (lavage) gp-340, a member of the scavenger receptor cysteine-rich protein family. Immunoblotting with antibodies that specifically recognized either lung gp-340 or the agglutinin confirmed that the salivary agglutinin was gp-340. Immunoblotting with an antibody specific to the sialyl Le(x) carbohydrate epitope detected expression on the salivary but not the lung glycoprotein, possible evidence of different glycoforms. The salivary agglutinin also interacted with Helicobacter pylori, implicated in gastritis and peptic ulcer disease, Streptococcus agalactiae, implicated in neonatal meningitis, and several oral commensal streptococci. These results identify the salivary agglutinin as gp-340 and suggest it binds bacteria that are important determinants of either the oral ecology or systemic diseases.

Separate oligosaccharide determinants mediate interactions of the low-molecular-weight salivary mucin with neutrophils and bacteria.

The low-molecular-weight human salivary mucin (MG2) coats oral surfaces, where it is in a prime location for governing cell adhesion. Since oligosaccharides form many of the interactive facets on mucin molecules, we examined MG2 glycosylation as it relates to the molecule's adhesive functions. Our previous study of MG2 oligosaccharide structures showed that the termini predominantly carry T, sialyl-T, Lewisx (Lex), sialyl Lex (sLex), lactosamine, and sialyl lactosamine determinants [Prakobphol, A., et al. (1998) Biochemistry 37, 4916-4927]. In addition, we showed that sLex determinants confer L-selectin ligand activity to this molecule. Here we studied adhesive interactions between MG2 and cells that traffic in the oral cavity: neutrophils and bacteria. Under flow conditions, neutrophils tethered to MG2-coated surfaces at forces between 1.25 and 2 dyn/cm2, i.e., comparable to the shear stress generated at the tooth surface by salivary flow ( approximately 0.8 dyn/cm2). MG2 was also found in association with neutrophils isolated from the oral cavity, evidence that the cells interact with this mucin in vivo. Since MG2 serves as an adhesion receptor for bacteria, the MG2 saccharides that serve this function were also identified. Seven of 18 oral bacteria strains that were tested adhered to MG2. Importantly, six of these seven strains adhered via T antigen, sialyl-T antigen, and/or lactosamine sequences. No adherence to Lex and sLex epitopes was detected in all the strains that were tested. Together, these results suggest that distinct subsets of MG2 saccharides function as ligands for neutrophil L-selectin and receptors for bacterial adhesion, a finding with interesting implications for both oral health and mucin function.

Human low-molecular-weight salivary mucin expresses the sialyl lewisx determinant and has L-selectin ligand activity.

Previously we showed that the low-molecular-weight mucin (MG2, encoded by MUC7), a major component of human submandibular/sublingual saliva, is a bacterial receptor that coats the tooth surface. Here we tested the hypothesis that the structure of its carbohydrate residues contains important information about its function. Purified MG2 (Mr 120 000) was digested with trypsin, and the resulting Mr 90 000 fragment, which carried primarily O-linked oligosaccharides, was subjected to reductive beta-elimination. The released oligosaccharides were characterized by using nuclear magnetic resonance spectroscopy and mass spectrometry. Of the 41 different structures we detected, the most prominent included NeuAcalpha2-->3Galbeta1-->3GalNAc-ol (sialyl-T antigen), Galbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-->6(Galbeta1 -->3)GalNAc-ol [type 2 core with Lewisx (Lex) determinant], and NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6(Galbeta1--> 3) GalNAc-ol [type 2 core with sialyl Lex (sLex) determinant]. We also detected di-, tri-, and pentasaccharides with one sulfate group. Lex, sLex, and related sulfated structures are ligands for selectins, adhesion molecules that mediate leukocyte trafficking. Therefore, we investigated whether MG2 was a selectin ligand. In an enzyme-linked immunosorbent assay, L-selectin chimeras interacted with immobilized MG2 in a Ca2+-dependent manner. L-Selectin chimeras also bound to MG2 immobilized on nitrocellulose. Together, these results suggest that the saccharides that MG2 carries could specify some of its important functions, which may include mediating leukocyte interactions in the oral cavity.

Palmitoyl carnitine, a lysophospholipase-transacylase inhibitor, prevents Candida adherence in vitro.

Candida adherence is poorly understood. The results of this study indicate that interactions of Candida with the lyso-forms of phospholipids may be one important attachment mechanism. C. tropicalis and C. albicans adhered to purified lysophospholipids immobilized on microtiter wells, as well as to a human laryngeal epidermoid carcinoma (HEp-2) cell line. Adherence to both lysophospholipids and HEp-2 cells was significantly reduced by palmitoyl carnitine, a lysophospholipase-transacylase inhibitor. Over time there was a positive correlation between Candida adherence and its transacylase activity. The data suggest that palmitoyl carnitine interferes with Candida adherence to lysophospholipids and the HEp-2 cell line by blocking the interaction between the Candida-associated transacylase enzyme receptor site and its lysophospholipid substrate ligand.

PRB1, PRB2, and PRB4 coded polymorphisms among human salivary concanavalin-A binding, II-1, and Po proline-rich proteins.

Six closely linked PRP (proline-rich protein) genes code for many salivary PRPs that show frequent length and null variants. From determined protein sequences and DNA sequence analysis of variant alleles, we here report the coding and molecular basis for Con (concanavalin A-binding) and Po (parotid "o") protein polymorphisms. The Con1 glycoprotein is encoded in exon 3 of a PRB2 allele (PRB2L CON1+) with a potential N-linked glycosylation site. Because of a probable gene conversion encompassing > or = 684 bp of DNA, the "PRB2-like" Con2 glycoprotein is encoded in exon 3 of a PRB1 allele (PRB1M CON2+) with a potential glycosylation site. The PmF protein is also encoded in the PRB1M CON2+ allele, thus explaining the previously reported association between Con2 and PmF proteins. A PRB2L CON1 allele contains a single nt missense change [TCT(Ser)-->CCT (Pro)] that abolishes the potential N-linked glycosylation site (NKS-->NKP) in the Con1 protein, and this explains the Con- type. The Po protein and a glycoprotein (II-1) are encoded in the PRB4 gene, and both proteins are absent in the presence of a mutation in the PRB4M PO- allele that contains a single nt change (G--C) at the +1 invariant position of the intron 3 5'donor splice site. The genetically determined absence of the II-1 glycoprotein leads to altered in vitro binding of Streptococcus sanguis 10556 to salivary proteins, which suggests a biological consequence for null mutations of the PRB4 gene.

Quantifying the strength of bacterial adhesive interactions with salivary glycoproteins.

We adapted an assay that has been used to estimate the strength of eukaryotic cell-cell and cell-extracellular matrix adhesive interactions (McClay et al., 1981) to quantify the strength of bacterial (streptococci, fusobacteria, actinomyces) interactions with salivary receptors. Bacteria are centrifuged onto human submandibular/sublingual or parotid-saliva-coated microtiter wells. Plates are sealed with pressure-sensitive, double-sided tape which allows them to be inverted and centrifuged again. The force required to remove the bacteria from the coated wells is a direct measure of the adhesive strength of the interaction(s) being disrupted. The bacteria-saliva adhesive forces we detected ranged from 1.6 x 10(-8) dynes (Streptococcus sanguis 72-40) to > 1.1 x 10(-7) dynes (Actinomyces viscosus T14 V). These forces were in the range to withstand the shear stress produced by salivary flow, which we calculated as approximately 6.1 x 10(-7) dynes.

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

Cytotrophoblasts are the specialized epithelial cells of the placenta. During the first trimester, a subpopulation of chorionic villus cytotrophoblasts differentiates along an invasive pathway and penetrates the maternal endometrium, decidua and spiral arterioles. Cytotrophoblast invasiveness declines rapidly during the second half of gestation. Isolated cytotrophoblasts of different gestational ages retain this differential invasiveness in culture. To determine whether the properties of integrin receptors for extracellular matrix molecules differ between invasive and non-invasive cytotrophoblasts, detergent extracts of isolated cytotrophoblasts of different gestational ages, and of first-trimester villous fibroblasts, were immunoprecipitated with subunit-specific anti-beta 1 integrin antibodies. Striking alterations in electrophoretic mobility were observed in beta 1 integrins from first-trimester cytotrophoblasts, as compared with those from term cytotrophoblasts or first-trimester villous fibroblasts, suggesting a cell-type-specific, temporally regulated alteration in glycosylation. Treatment of total first-trimester cytotrophoblast beta 1 integrins or the isolated alpha 5/beta 1 fibronectin receptor with endo-beta-galactosidase restored electrophoretic mobility to control levels, suggesting the presence of polylactosamine-bearing oligosaccharides. Further analysis by enzyme digestion and lectin-affinity chromatography suggested that they consisted of at least three antennae and short, sialylated lactosamine units. These oligosaccharides did not affect the affinity of the first-trimester cytotrophoblast fibronectin receptor for fibronectin. However, this receptor bound more strongly to wheat germ agglutinin than control fibronectin receptor and resisted elution by high concentrations of sugar hapten, requiring ionic detergent for removal. These results suggested that the altered glycosylation affected the conformation of the fibronectin receptor.

Specific adherence of Candida tropicalis to lysophospholipids.

Candida species are usually commensal organisms, but they become invasive when the host is immunocompromised. Mechanisms by which these organisms adhere to, colonize, and then invade host tissues are poorly understood. To detect potential host receptors, members of a lipid library were chromatographically separated and then overlaid with Candida tropicalis; components to which the organisms bound were visualized by autoradiography. In initial experiments no interactions with either glycolipids or intact phospholipids were detected. However, lysophospholipids supported adherence of C. tropicalis but not Saccharomyces cerevisiae. These results were confirmed by a second assay; C. tropicalis adhered to certain lysophospholipids, but not intact phospholipids, that were immobilized on microtiter plates. Using [14C]-1-palmitoyl-sn-glycero-3-phosphocholine, we showed that C. tropicalis adherence is accompanied by rapid conversion of the labeled lipid to a number of compounds. Thus, the interaction of C. tropicalis with lysophospholipids results in significant changes in both the organism and the lysophospholipid to which it binds. We hypothesize that this interaction could be an important component of the infection process.

PRB3 null mutations result in absence of the proline-rich glycoprotein Gl and abolish Fusobacterium nucleatum interactions with saliva in vitro.

The glycosylated proline-rich glycoprotein (Gl or PRG), a product of the PRB3 gene, is a major constituent of human parotid saliva. Important functions proposed for Gl include acting as a bacterial receptor. The Gl proteins of several subjects were typed by two polyacrylamide gel electrophoresis (PAGE) systems: acid-lactate PAGE followed by staining with the periodic acid-Schiff reagent and sodium dodecyl sulfate-PAGE followed by electrophoretic transfer and staining with amido black or concanavalin A. The results showed one subject who apparently lacked Gl. The four exons, including splice junctions, for both PRB3 alleles of this subject were completely sequenced. Unexpressed (null) mutations were detected with an identical C nucleotide insertion in the same coding region of exon 3 of both alleles. This C nucleotide insertion leads to a frameshift with a premature termination codon that probably results in markedly reduced or absent PRB3 gene expression. We then used a nitrocellulose blot overlay assay to assay the bacterial receptor activity of parotid saliva from the PRB3null subject. No interactions with Fusobacterium nucleatum, shown previously to interact selectively with Gl, were detected. Together, these results suggest that this subject does not express the PRB3 gene and that one of the consequences is an altered ability to interact with a bacterium known to colonize the oral cavity.

The high-molecular-weight human mucin is the primary salivary carrier of ABH, Le(a), and Le(b) blood group antigens.

Because many bacteria interact with the carbohydrate portions of receptor molecules, factors controlling glycosylation probably influence the ability of salivary components to mediate bacterial adherence/clearance. Important sources of diversity in glycosylation are the ABO, secretor, and Lewis genes, which code for glycosyltransferases that add specific sugar sequences to the termini of carbohydrate chains of glycolipids and glycoproteins. We identified, by Western blotting, salivary glycoproteins carrying the ABH and Le(a) or Le(b) antigens. Samples of whole, unstimulated saliva were obtained from 19 subjects whose blood group was determined by agglutination of red blood cells with specific antisera. After centrifugation, the samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose. Glycoproteins carrying blood group antigens were identified by staining the blot with monoclonal antisera specific for the A, B, H, Le(a), or Le(b) antigens. The most intensely staining component from all the samples migrated at the same position as the high-molecular-weight mucin. Saliva samples from the nonsecretors contained only the Le(a) antigen. Samples from the secretors contained one or more of the ABH antigens and, variably, the Le(b) antigen. In all cases, the salivary blood group antigens corresponded to those found on the red blood cells of the same subject. The functional consequences of the expression of blood group antigens on the high-molecular-weight mucin are not known, but their presence could modulate the adherence of certain oral microorganisms that interact preferentially with this molecule.

Adherence of oral streptococci to salivary glycoproteins.

We used an overlay method to study the ability of human salivary glycoproteins to serve as receptors for several strains of streptococci that colonize the oral cavity. Parotid and submandibular-sublingual salivas were collected as ductal secretions, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. The resulting blots were overlaid with [35S]methionine-labeled bacteria, and salivary components to which the bacteria bound were detected by autoradiography. Potential glycoprotein receptors were identified for 8 of the 16 strains tested. In three cases (Streptococcus sanguis 72-40 and 804 and Streptococcus sobrinus OMZ176), highly specific interactions with a single salivary component were detected. Removal of sialic acid residues from the low-molecular-weight salivary mucin prevented adherence of one of these strains (S. sanguis 72-40), suggesting that this saccharide either mediates binding or is a critical component of the receptor site. In the remaining five strains (Streptococcus gordonii G9B and 10558, S. sanguis 10556, and Streptococcus oralis 10557 and 72-41), interactions with multiple salivary components, including the low-molecular-weight salivary mucin, highly glycosylated proline-rich glycoproteins, and alpha-amylase, were detected. These results suggest that some oral streptococci can bind specifically to certain of the salivary glycoproteins. The interactions identified may play an important role in governing bacterial adherence and clearance within the oral cavity.

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.

Effect of plasma on composition of human enamel and cementum pellicle.

Slabs of human enamel and cementum were incubated with plasma alone or with various mixtures of plasma and saliva. Proteins and glycoproteins that adsorbed to the surface of the slabs in 0 to 60 min were labeled by lactoperoxidase-catalyzed 125I-iodination and by mild periodate oxidation followed by NaB3H4 reduction. The labeled components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography or fluorography. From plasma alone, a 58 and a 66 kDa protein (probably albumin) were adsorbed to the enamel surface in relatively equal amounts, but no 125I-labeled components were detected on the cementum surface in the absence of saliva. Adding 10% saliva to the incubation mixture promoted the adsorption of the 58 and 66 kDa components to cementum. In addition, another set of proteins, including components of 44, 47, 29, and 25 kDa, was adsorbed to both cementum and enamel in the presence of saliva. These six proteins were the major 125I-labeled species in all of the pellicles formed from mixtures of plasma and saliva. The electrophoretic mobility of the major 120 and 140 kDa 3H-labeled sialoglycoproteins adsorbed to both cementum and enamel was similar to that of the low-molecular-weight mucin of submandibular/sublingual saliva.

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.

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.

External radiolabelling of components of pellicle on human enamel and cementum.

Enamel and cementum pellicles form by different adsorption of salivary and serum components to the tooth surface. The authors compared the constituents of surface pellicle formed on human enamel and cementum under three conditions: (1) natural pellicle, present on extracted teeth, which was formed by prolonged exposure to human salivary and serum components in vivo; (2) short-term in-vivo pellicle, formed by exposing enamel and cementum slabs to the oral environment for 0-60 min; (3) in-vivo pellicle, formed by incubating enamel and cementum slabs in a 1:1 mixture of parotid and submandibular/sublingual saliva for 0-60 min. Pellicle composition was characterized by external radiolabelling techniques specific for exposed carbohydrate (sialic acid and galactose) and amino-acid (tyrosine) residues. There were differences between cementum and enamel in the electrophoretic profiles of natural-pellicle components; notably, a major 180 kda 3H-labelled sialoglycoprotein, unique to the cementum pellicle, had the same electrophoretic mobility as the low-molecular-weight mucin from human submandibular/sublingual saliva. After alkaline-borohydride treatment, 3H-labelled natural-pellicle oligosaccharides chromatographed in the di- to tetrasaccharide region of a Bio-Gel P-2 column. The most prominently labelled components of short-term enamel and cementum pellicles in vivo and in vitro had the same electrophoretic mobility as the low-molecular-weight salivary mucin. The pellicle components formed in vitro, unlike those formed for the same period of time in vivo, were rapidly desorbed from the cementum, but not from the enamel surface. We conclude that: (1) external labelling techniques are useful for obtaining a profile of pellicle components; (2) submandibular/salivary mucins are major constituents of salivary pellicles on tooth surfaces; (3) glycoproteins that carry low-molecular-weight, sialic-acid-containing saccharides are important determinants of pellicle surface properties [corrected].

Oligosaccharide structures of the low-molecular-weight salivary mucin from a normal individual and one with cystic fibrosis.

Studies were begun to compare the oligosaccharide structures of the low-molecular-weight mucin purified from the submandibular-sublingual saliva of a normal individual with that from one with cystic fibrosis. Following alkaline/borotritide cleavage, neutral and sialic acid-containing chains were purified by a combination of gel filtration, paper chromatography, and high-voltage paper electrophoresis. Oligosaccharides ranged in size from a disaccharide to a heptasaccharide. Approximately 80% of the oligosaccharides were GalB1, 3GalNAc; Fuc alpha 1,2GalB1,3GalNAc; and NeuAc alpha 2,3GalB1, 3GalNAc. The other structures were Fuc alpha 1,2GalB1,4(Fuc alpha 1,3)GlcNAcB1,6(GalB1,3)GalNAc or GalB1,4(Fuc alpha 1,3)GlcNAcB1,6(Fuc alpha 1,2GalB1,3)GAlNAc; GalB1,4(Fuc alpha 1,3)GlcNAcB1,6(NeuAc alpha 2,3GalB1,3)GalNAc; and Fuc alpha 1,2GalB1,4(Fuc alpha 1,3)GlcNAcB1,6 (NeuAc alpha 2,3GalB1,3)GalNAc. There were no apparent qualitative differences in the neutral and sialic acid-containing units recovered from the normal and cystic fibrosis samples.

Characterization of serological cross-reactivity between polysaccharide antigens of Streptococcus mutans serotypes c and d.

Immunological assays with antisera prepared against purified Streptococcus mutans serotype c polysaccharide demonstrated that a cross-reacting determinant on c polysaccharide reacted with the wall-associated rhamnose-glucose polysaccharide from S. mutans serotype d. Studies with 60 antisera prepared against chemostat cultures of S. mutans Ingbritt (c) demonstrated that the rhamnose-glucose polysaccharide cross-reactive determinant was consistently expressed on c antigen under a variety of growth conditions.