In vivo binding of the salivary glycoprotein EP-GP (identical to GCDFP-15) to oral and non-oral bacteria detection and identification of EP-GP binding species.

Extra Parotid Glycoprotein (EP-GP) is a glycoprotein isolated from human saliva, having homologues in several other body fluids. The biological role of EP-GP and its homologues is unknown. Recently, EP-GP was shown to bind in vitro to the bacterium Streptococcus salivarius HB. In contrast, no binding to a number of other oral microorganisms could be demonstrated. In the present study we have determined whether binding of EP-GP to bacteria occurs in vivo in saliva and in other EP-GP containing body fluids. Therefore the presence of EP-GP on bacteria in vivo was determined by analyzing oral, skin and ear floras by confocal fluoresence microscopy using specific antibodies. About 12% of the in vivo oral flora had EP-GP present on their surface, while approximately 5% of the bacteria from ear canal or skin was positive for EP-GP. IgA was detected on approximately 65% of the salivary bacteria, whereas the high-molecular weight mucin (MG1) and cystatin C were not detectable on any oral bacterium. Using a replica-plate assay, a number of EP-GP binding strains in saliva were isolated and identified as Gemella haemolysans, Gemella morbillorium, Streptococcus acidominimus, Streptococcus oralis, Streptococcus salivarius and Streptococcus parasanguis. Bacteria from the ear canal and skin bacteria were identified as Staphylococcus hominis. It is concluded that EP-GP is selectively bound in vivo to several oral and non-oral bacterial species.

EP-GP and the lipocalin VEGh, two different human salivary 20-kDa proteins.

Two salivary 20-kDa proteins [the human lipocalin Von Ebner's gland protein (VEGh) and extraparotid glycoprotein (EP-GP)] show several remarkable similarities and differences. The latter is identical to secretory actin-binding protein (SABP), gross cystic disease fluid protein-15 (GCDFP-15), prolactin-induced protein (PIP), and 17-kDA CD4-binding glycoprotein (gp17). Much is known about the distribution, localization, biochemical characteristics, and molecular biology of these two proteins, yet there are only few clues about their functions.

Binding of human high-molecular-weight salivary mucins (MG1) to Hemophilus parainfluenzae.

In human saliva, two different mucin populations can be distinguished, viz., high-molecular-weight mucins (MG1, mol. wt > 1 x 10(6)) and low-molecular-weight mucins (MG2, mol. wt approximately 125 kD). The carbohydrate moiety of MG1 displays a wide spectrum of oligosaccharide structures, varying in composition, length, branching, and acidity. The biological significance of the heterogeneity in carbohydrate structures of mucins is unclear. The present investigation focused on the question whether MG1, because of its diverse carbohydrate side-chain population, can bind to a large variety of oral micro-organisms. A replica plate technique, in combination with immunochemical detection with monoclonal antibodies against MG1, was used to screen in vivo human oral microflora for the presence of micro-organisms which could bind the high-molecular-weight salivary mucin MG1. Binding to purified MG1 was established for Hemophilus (para)influenzae species, whereas other species, including Streptococcus and Staphylococcus, were negative. MG1 binding to Hemophilus parainfluenzae could be abolished by protease treatment of MG1. In contrast, periodate acid treatment, partial deglycosylation, or addition of monosaccharides did not affect MG1 binding to H. parainfluenzae, indicating that MG1 carbohydrate side-chains were not directly involved in the binding. The binding was pH-dependent, showing an increase when the pH was lowered from 8.0 to 4.0. These data indicate that MG1 can be bound in a selective manner by Hemophilus spp. and suggest that the 'naked' unglycosylated polypeptide moiety of MG1 is involved in its binding to Hemophilus parainfluenzae.

Biochemical composition of human saliva in relation to other mucosal fluids.

This paper describes several salivary components and their distribution in other mucosal secretions. Histatins are polypeptides which possess exceptional anti-fungal and anti-bacterial activities, but are nevertheless present only in saliva. Proline-rich proteins (PRPs) are members of a closely related family, of which the acidic PRPs are found solely in saliva, whereas the basic PRPs are also found in other secretions. Mucins are a group of glycoproteins that contribute to the visco-elastic character of the mucosal secretions. Despite the similarities in their structure and behavior, mucins have distinct tissue distributions and amino acid sequences. Other salivary proteins are present in one or more mucosal secretions. Lysozyme is an example of a component belonging to an ancient self-defense system, whereas secretory immunoglobulin A (sIgA) is the secreted part of a sophisticated adaptive immune system. Cystatins are closely related proteins which belong to a multigene family. Alpha-Amylase is a component that is believed to play a specific role in digestion, but is nevertheless present in several body fluids. Kallikrein and albumin are components of blood plasma. But whereas albumin diffuses into the different mucosal secretions, kallikrein is secreted specifically by the mucosal glands. The presence of these proteins specifically in saliva, or their distribution in other mucosal secretions as well, may provide important clues with respect to the physiology of those proteins in the oral cavity.

Identity of human extra parotid glycoprotein (EP-GP) with secretory actin binding protein (SABP) and its biological properties.

In this paper the identity of the salivary protein EP-GP (extra-parotid glycoprotein) is reported, also apparent in other human secretions. Immunochemical and biochemical analysis demonstrated that EP-GP is similar to the secretory actin-binding protein (SABP), also known as gross cystic disease fluid protein-15 (GCDFP-15) and prolactin-inducible protein (PIP). The molecular mass and charge microheterogeneity of EP-GP, also observed for SABP, was shown to be predominantly caused by the carbohydrate moiety. In addition, evidence was given that EP-GP is not related to the lipocalin Von Ebner's gland protein (human; VEGh). The biological significance of EP-GP and its homologues is not clear. EP-GP bound to actin and fibrinogen as described for SABP and GCDFP-15. However, the affinity for these proteins does not appear to have any direct physiological role in the mucosal secretions. On the other hand, EP-GP binds to several bacteria. By electron microscopy the ultrastructural localization is demonstrated of EP-GP to the cell wall of both Streptococcus salivarius HB and its cell appendage-lacking mutant Streptococcus salivarius HB-C12. Concerning this finding we hypothesize on the possible functional aspects of this enigmatic protein EP-GP.

Interaction of the salivary glycoprotein EP-GP with the bacterium Streptococcus salivarius HB.

The interaction of the human salivary glycoprotein EP-GP with a number of oral bacterial species, following incubation with human whole saliva, has been investigated. EP-GP could be detected with a specific monoclonal antibody, by means of ELISA or by electrophoresis in combination with Western Transfer. The results indicated that EP-GP is bound only by Streptococcus salivarius, and not by the other tested strains of bacteria, Actinomyces viscosus, A. naeslundii, Actinobacillus actinomycetemcomitans, Bacteroides fragilis, S. gordonii, S. oralis, S. sanguis, S. mitis, S. mutans, S. sobrinus, S. rattus, S. constellatus, and S. anginosus. Binding of EP-GP to S. salivarius is mediated by a protein-protein interaction, which was found to be pH-dependent with a maximum binding between pH 5 and 6. For further characterization of the binding of EP-GP to S. salivarius, four mutants were tested, each of them lacking different cell wall antigens. EP-GP was bound to all mutants in amounts comparable with the wildtype, in spite of the different surface antigen compositions. We were able to identify a 27-kD EP-GP binding protein, by extraction of S. salivarius-cell wall antigens and electrophoretic techniques. In addition to EP-GP, S. salivarius also bound two other salivary proteins, namely, secretory IgA and low-molecular-weight mucin (MG-2).

Changes in saliva of epileptic patients.

Unstimulated whole saliva samples of 27 indoor epileptic patients were studied on their protein composition using biochemical and immunochemical methods. A number of salivary proteins appeared at least partially to be hydrolyzed. In a number of saliva samples the concentration of carbohydrate-containing isoenzymes of amylase was reduced. In addition, the concentration of the 20 kD glycoprotein EP-GP was reduced by 60%. Sialic acid, the terminal sugar of the glycoproteins and mucins, was released for about 50% and in three salivas even nearly completely. Moreover, sialic acid- and fucose-containing epitopes could hardly be detected by monoclonal antibodies to human salivary mucins. As a consequence of this hydrolytic breakdown the saliva mediated aggregation of two S. sanguis strains had been reduced. In contrast, the aggregation of S. oralis had been maintained.

Detection of proteins related to a salivary glycoprotein (EP-GP). Concentrations in human secretions (saliva, sweat, tears, nasal mucus, cerumen, seminal plasma).

With a highly specific monoclonal antibody against a previously isolated and characterized human salivary 19-20-kDa glycoprotein, designated as extra-parotid glycoprotein [Rathman et al. (1989) J. Biol. Buccale 17, 199-208], a common epitope was detected on proteins in several excretory human body fluids. With a quantitative ELISA the EP-GP epitope was measured in widely different concentrations in several secretory human body fluids in the descending order of seminal plasma much greater than tears approximately nasal mucus approximately sweat much greater than saliva. Crossreactivity was also observed in cerumen but not in milk, cerebrospinal fluid, blood plasma and urine. The relative amount of EP-GP in the positively reacting secretions was however, in the same order in each fluid per mg of protein on an average of 1% of the total protein amount. The EP-GP-epitope bearing proteins found in the various human secretions were further characterized by means of electrophoresis and immunoblotting. The molecular masses and the isoelectric points of the proteins in the different secretions display strong resemblance to values found for the salivary glycoprotein EP-GP (molecular masses 19 and 20 kDa; pI values between 4.8 and 5.4). All these findings point to the presence of proteins related to EP-GP in human secretions other than saliva.

Heparan sulfate proteoglycan from human tubular basement membrane. Comparison with this component from the glomerular basement membrane.

Heparan sulfate proteoglycan (HSPG) was extracted from human tubular basement membrane (TBM) with guanidine and purified by ion-exchange chromatography and gel filtration. The glycoconjugate was sensitive to heparitinase and resistant to chondroitinase ABC, had an apparent molecular mass of 200-400 kDa and consisted of 70% protein and 30% glycosaminoglycan. The amino acid composition was characterized by its high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulfonic acid yielded core proteins of 160 and 110 kDa. The heparan sulfate (HS) chains obtained after alkaline NaBH4 treatment had a molecular mass of about 18 kDa. Results of heparitinase digestion and HNO2 treatment suggest a clustering of sulfate groups in the distal portion of the HS side chains. These chemical data are comparable to those obtained previously on glomerular basement membrane (GBM) HSPG (Van den Heuvel et al. (1989) Biochem. J. 264, 457-465). Peptide patterns obtained after trypsin, clostripain or V8 protease digestion of TBM and GBM HSPG preparations showed a large similarity. Polyclonal antisera and a panel of monoclonal antibodies raised against both HSPG preparations and directed against the core protein showed complete cross-reactivity in ELISA and on Western blots. They stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies on human kidneys. Based on these biochemical and immunological data we conclude that HSPGs from human GBM and TBM are identical, or at least very closely related, proteins.