Substitution of bovine dentine sialoprotein with chondroitin sulfate glycosaminoglycan chains.

Dentine sialoprotein (DSP) represents 5-8% of all non-collagenous proteins present in the tooth, but, together with dentine phosphoprotein, has been shown to be vital for correct tooth formation. Recently, the existence of a highly glycosylated form of porcine DSP has been reported and it was shown to possess glycosaminoglycan (GAG) chains. The current investigation confirms that this is also the case for bovine DSP and has further characterized these carbohydrates. Dentine sialoprotein was purified from bovine dentine extracts by anion exchange chromatography and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and mass spectroscopy. An increase in molecular mass was observed, from 120 kDa to greater than 250 kDa, with a corresponding rise in anionic strength. Cellulose acetate electrophoresis and western blotting indicated the presence of chondroitin sulfate GAG chains within these dentine fractions. Further examination using sequential digestion with chondroitinase AC and N-glycosidase cleaved the samples first to 95 kDa and then to 80 kDa, respectively, confirming a high level of glycosylation. These results support the classification of bovine DSP as a proteoglycan, and that the carbohydrate substitutions may contribute to the functional properties of DSP.

Three SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) enhance factor H's cofactor activity enabling MCP-like cellular evasion of complement-mediated attack.

Previously we have shown that two members of the newly named SIBLING (small integrin-binding ligand, N-linked glycoproteins) family of proteins, bone sialoprotein, and osteopontin, bound first to a cell surface receptor and then to complement Factor H thereby blocking the lytic activity of the alternative pathway of complement. Another member of this family, dentin matrix protein 1, is shown in this paper to be very similar to osteopontin in that it can bind strongly to Factor H (K(a) approximately 1 nm) and block the lytic activity through either the vitronectin receptor (alpha(V)beta(3) integrin) or CD44. Binding of Factor H to SIBLING localized to the cells surface was demonstrated by fluorescence-activated cell sorting. Extensive overlapping fragment analyses suggests that both dentin matrix protein 1 and osteopontin interact with cell surface CD44 through their amino termini. Similar fragments of bone sialoprotein, like the intact protein, did not functionally interact with CD44. All three proteins are shown to act in conjunction with Factor I, a serum protease that, when complexed to appropriate cofactors, stops the lytic pathway by digesting the bound C3b in a series of proteolytic steps. These results show that at least three members of this family confer membrane cofactor protein-like activity (MCP or CD46) upon cells expressing RGD-binding integrins or CD44. The required order of the assembly of the complex suggests that this cofactor activity is limited to short diffusional distances.

A novel rat dentin mRNA coding only for dentin sialoprotein.

Dentin sialoprotein (DSP) is a major glycoprotein present in the mineralized dentin matrix that is expressed mainly by young and mature odontoblasts. Mutations in the DSP coding regions are linked to Dentinogenesis imperfecta I and II. indicating the importance of DSP in tooth formation. Previous studies have identified multiple mRNA transcripts in dentin that code for both DSP and phosphophoryns (PPs). Using reverse transcriptase-polymerase chain reaction (RT-PCR) to characterize these mRNA transcripts, we have identified a cDNA that codes for DSP, but not PP. This cDNA codes for a protein with 324 amino acids, 303 amino acids being identical to the published rat DSP sequence. However, the subsequent 21 amino acids are unique to this cDNA. Based on the coding sequence, the core protein is predicted to have a pI=4.24, a net charge of -34, and to contain four potential N-glycosylation sites and six potential sites for phosphorylation by casein kinase. That the corresponding mRNA was present in day 5 molar tooth germs was confirmed using RNA protection assays. These data, therefore, identify a novel transcript in rat tooth germs that codes only for DSP (designated as DSPII).

Identification of endoglycan, a member of the CD34/podocalyxin family of sialomucins.

CD34 and podocalyxin are structurally related sialomucins, which are expressed in multiple tissues including vascular endothelium and hematopoietic progenitors. These glycoproteins have been proposed to be involved in processes as diverse as glomerular filtration, inhibition of stem cell differentiation, and leukocyte-endothelial adhesion. Using homologies present in the cytoplasmic tails of these proteins, we have identified a novel member of this family, which we designate endoglycan. This protein shares a similar overall domain structure with the other family members including a sialomucin domain, but also possesses an extremely acidic amino-terminal region. In addition, endoglycan contains several potential glycosaminoglycan attachment sites and is modified with chondroitin sulfate. Endoglycan mRNA and protein were detected in both endothelial cells and CD34(+) bone marrow cells. Thus, CD34, podocalyxin, and endoglycan comprise a family of sialomucins sharing both structural similarity and sequence homology, which are expressed by both endothelium and multipotent hematopoietic progenitors. While the members of this family may perform overlapping functions at these sites, the unique structural features of endoglycan suggest distinct functions for this molecule.

Chemokines: what chemokine is that?

The discovery of a new and unusual member of the chemokine family illustrates the importance of chemoattractant diversity in the regulation of leukocyte movement through the body. The chemokines are now divisible into four clearly defined subgroups on the basis of structural and functional properties.

Intracellular IL-1 receptor antagonist promoter: cell type-specific and inducible regulatory regions.

The objective of these studies was to examine the molecular mechanisms involved in transcriptional regulation of the gene for the intracellular structural variant of the IL-1 receptor antagonist (icIL-1Ra) molecule. By reverse transcription-PCR analysis, constitutive expression of endogenous icIL-1Ra mRNA was observed in the epithelial cell lines A431 and HT-29, but not in the macrophage cell lines RAW 264.7 and U937, or in the lymphocyte cell lines Raji and Jurkat. However, icIL-1Ra mRNA expression was observed in response to stimulation with LPS in RAW 264.7 cells and to PMA and LPS in U937 cells. To examine the mechanisms of transcriptional regulation, 4.5 kb of the 5' flanking sequence was isolated from the human icIL-1Ra gene, sequenced, cloned into a luciferase expression vector (pIC4525.Luc), and examined in transfection studies. The pIC4525.Luc construct exhibited a pattern of expression in epithelial and macrophage cell lines similar to that of the endogenous icIL-1Ra gene. To obtain a generalized map of cell type-specific and inducible cis-acting DNA elements, nested 5' deletional mutants of the icIL-1Ra promoter were constructed. Results from transfection studies with these icIL-1Ra promoter/luciferase fusion constructs indicated that constitutive expression in epithelial cells was under the control of three positively acting regions located between bases -4525 to -1438, -288 to -156, and -156 to -49. In contrast, basal expression of pIC4525.Luc in transfected but unstimulated RAW 264.7 cells was under the control of a weak inhibitory region located between bases -4525 to -1438 and a strong positive element between -156 and -49. LPS induction of icIL-1Ra transcription in RAW 264.7 cells was regulated by strong positively acting DNA regions between bases -1438 to -909 and -156 to -49. In summary, the proximal region of the icIL-1Ra promoter, between bases -156 to -49, contains positive cis-acting elements that are needed for expression in both epithelial and monocyte cell lines. However, our results indicate that the ability of this proximal promoter region to control expression is strongly influenced, both positively and negatively, by other upstream cis-acting elements in a cell type-specific manner.

Characterization of porcine intestinal receptors for the K88ac fimbrial adhesin of Escherichia coli as mucin-type sialoglycoproteins.

We have previously identified two K88ac adhesion receptors (210 and 240 kDa) which are present in membrane preparations from adhesive but not nonadhesive porcine intestinal brush border cells; these adhesin receptors are postulated to be important determinants of the susceptibility of pigs to K88ac+ enterotoxigenic Escherichia coli infections (A.K. Erickson, J.A. Willgohs, S.Y. McFarland, D.A. Benfield, and D.F. Francis, Infect. Immun. 60:983-988, 1992). We now describe a procedure for the purification of these two receptors. Receptors were solubilized from adhesive intestinal brush border vesicles using deoxycholate and were purified by gel filtration chromatography on Sepharose CL-4B and then by hydroxyapatite chromatography. Amino acid compositional analyses indicated that the two receptors have similar amino acid compositions. The most distinguishing characteristic of both receptors is a high percentage of threonine and proline residues. Neuraminidase treatment caused the K88ac adhesin receptors to migrate with a slower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, indicating that these receptors are sialoglycoproteins. Results from lectin-binding studies indicated that the receptors contain O-linked oligosaccharides composed of galactosyl (beta-1,3)N-acetylgalactosamine, alpha-linked fucose, galactosyl(beta-1,4)N-acetylglucosamine, sialic acid, galactose, and N-acetylgalactosamine. Collectively, these characteristics indicate that the K88ac adhesin receptors are mucin-type sialoglycoproteins.

Multiple forms of SppI (secreted phosphoprotein, osteopontin) synthesized by normal and transformed rat bone cell populations: regulation by TGF-beta.

Metabolic labeling has revealed that rat bone cell populations in culture synthesize several forms of the secreted phosphoprotein, SppI. Most cell populations produced two major [32PO4]-labeled forms that behaved anomolously on SDS-PAGE migrating at 60 kDa and 56 kDa on 10% gels and 55 kDa and 44 kDa on 15% gels. Minor forms of intermediate sizes were also resolved. In normal bone cells the 60 kDa form was predominant and was the only form produced by the clonal bone cell line, RCA 11, whereas the 56 kDa a form predominated in the transformed bone cell line, ROS 17/2.8. In all populations [35S]-methionine-labeling revealed SppIs at approximately 60 kDa but no 56 kDa form. Each form of SppI was specifically cleaved by thrombin which generated fragments of approximately 28 kDa. Transforming growth factor beta 1 increased SppI mRNA levels 3 to 6-fold within 24 h in the normal bone cells, but no increase occurred in the ROS 17/2.8 cells. The elevated expression of SppI was reflected in a selective increase in the synthesis of the [32PO4]-and [35S]-methionine-labeled 60 kDa SppIs.

Expression on blood cells of sialophorin, the surface glycoprotein that is defective in Wiskott-Aldrich syndrome.

Sialophorin, previously called gpL115, is the heavily sialylated surface protein that is defective in lymphocytes of Wiskott-Aldrich syndrome patients. Using the monoclonal antibody L10 as a probe, sialophorin expression was detected on isolated T lymphocytes and thymocytes, B cell lines, monocytes, neutrophils, and platelets, but not on erythrocytes, fibroblasts, and glioblastoma cells. This unusual distribution pattern suggests that sialophorin is expressed on all circulating cells except erythrocytes. Trace amounts of the sialophorin molecules on lymphocytes are incompletely sialylated, but significant amounts of the molecules on thymocytes are incompletely sialylated. The molecular form of sialophorin on T lymphocytes, thymocytes, and monocytes is the previously characterized species of apparent mol wt 115,000. A newly described sialophorin species of apparent mol wt 135,000 was found on neutrophils and platelets. The 115,000 lymphocyte/monocyte form and the 135,000 platelet/neutrophil form were shown to be substantially similar. The two forms have approximately the same content of sialylated O-linked carbohydrate units since both undergo the same atypical shift in electrophoretic mobility on desialylation. Both contain the epitope recognized by the monoclonal antibody L2 and the epitope recognized by L10 antibody. Moreover, evidence from another study indicates that the polypeptide portions are identical, cumulatively suggesting that 115,000 sialophorin and 135,000 sialophorin are identical except for the presence on the latter of additional neutral saccharide residues.

Structures of Miltenberger class I and II specific major human erythrocyte membrane sialoglycoproteins.

The N-terminal structures of the Miltenberger (Mi-) blood group class I and II specific human MN erythrocyte membrane sialoglycoproteins were determined by manual sequencing of tryptic glycopeptides and various secondary fragments. The Mi-I and Mi-II active glycoproteins were found to exhibit a threonine leads to methionine and threonine leads to lysine exchange, respectively, at position 28 which prevents N-glycosylation of asparagine 26. Due to the absence of the N-glycosidic oligosaccharide chain, the monomeric form of the Mi-I and Mi-II specific glycoproteins possesses a slightly increased sodium dodecyl sulfate/polyacrylamide gel electrophoretic mobility, in comparison to its normal counterpart. Serological studies suggest that antibodies, specific for Mi-I or Mi-II red cells, react with the structurally altered region of the MN glycoprotein.

Isolation and characteristics of human urinary sialoglycoproteins.

Three sialoglycoproteins (S1, S2, and S3) have been isolated from normal human urine by ultrafiltration, zone electrophoresis, gel chromatography, and ion-exchange chromatography. The average yields per litre of urine were 0.39 mg (S1), 0.40 mg (S2), and 1.9 mg (S3). The isolated substances were homogeneous on ultracentrifugation both at neutral and acid pH with sedimentation coefficients of 3.6 S (S1), 2.4S (S2), and 0.93S (S3). Equilibrium ultracentrifugation gave molecular weights of 77900 (S1), 37000 (S2), and 5300 (S3). All three components were rich in carbohydrate (64 to 76%) and contained 28 to 35% of sialic acid. Alkaline borohydride degradation of component S3 yielded two sialylated oligosaccharides which were partially characterized. The origin of the isolated substances is unknown. The molecular size of S3 (Stokes' radius of 2.0 nm) is compatible with passage from the blood by glomerular filtration whereas the size of S1 (Stokes' radius of 6.5 nm) would suggest a renal origin.