Glycosaminoglycan-binding Protein Research Articles

Predicting gene signature in breast cancer patients with multiple machine learning models

AimsThe aim of this study was to predict gene signatures in breast cancer patients using multiple machine learning models.MethodsIn this study, we first collated and merged the datasets GSE54002 and GSE22820, obtaining a gene expression matrix comprising 16,820 genes (including 593 breast cancer (BC) samples and 26 normal control (NC) samples). Subsequently, we performed enrichment analyses using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Disease Ontology (DO).ResultsWe identified 177 differentially expressed genes (DEGs), including 40 up-regulated and 137 down-regulated genes, through differential expression analysis. The GO enrichment results indicated that these genes are primarily involved in extracellular matrix organization, positive regulation of nervous system development, collagen-containing extracellular matrix, heparin binding, glycosaminoglycan binding, and Wnt protein binding, among others. KEGG enrichment analysis revealed that the DEGs were primarily associated with pathways such as focal adhesion, the PI3K–Akt signaling pathway, and human papillomavirus infection. DO enrichment analysis showed that the DEGs play a significant role in regulating diseases such as intestinal disorders, nephritis, and dermatitis. Further, through LASSO regression analysis and SVM-RFE algorithm analysis, we identified 9 key feature DEGs (CF-DEGs): ANGPTL7, TSHZ2, SDPR, CLCA4, PAMR1, MME, CXCL2, ADAMTS5, and KIT. Additionally, ROC curve analysis demonstrated that these CF-DEGs serve as a reliable diagnostic index. Finally, using the CIBERSORT algorithm, we analyzed the infiltration of immune cells and the associations between CF-DEGs and immune cell infiltration across all samples.ConclusionsOur findings provide new insights into the molecular functions and metabolic pathways involved in breast cancer, potentially aiding in the discovery of new diagnostic and immunotherapeutic biomarkers.

Revealing the Identity of Human Galectin-3 as a Glycosaminoglycan-Binding Protein.

Human galectin-3 (Gal-3) is a β-galactoside-binding lectin. This multitasking protein preferentially interacts with N-acetyllactosamine moieties on glycoconjugates. Specific hydroxyl groups (4-OH, 6-OH of galactose, and 3-OH of glucose/N-acetylglucosamine) of lactose/LacNAc are essential for their binding to Gal-3. Through hemagglutination inhibition, microcalorimetry, and spectroscopy, we have shown that despite being a lectin, Gal-3 possesses the characteristics of a glycosaminoglycan (GAG)-binding protein (GAGBP). Gal-3 interacts with sulfated GAGs [heparin, chondroitin sulfate-A (CSA), -B (CSB), and -C (CSC)] and chondroitin sulfate proteoglycans (CSPGs). Heparin, CSA, and CSC showed micromolar affinity for Gal-3, while the affinity of CSPGs for Gal-3 was much higher (nanomolar). Interestingly, CSA, CSC, and a bovine CSPG, not heparin and CSB, were multivalent ligands for Gal-3, and they formed reversible noncovalent cross-linked complexes with the lectin. Binding of sulfated GAGs to Gal-3 was completely inhibited when Gal-3 was preincubated with β-lactose. Cross-linking of Gal-3 by CSA, CSC, and the bovine CSPG was also reversed by β-lactose. These findings strongly suggest that GAGs primarily occupy the lactose/LacNAc binding site of Gal-3. Identification of Gal-3 as a GAGBP should help to reveal new functions of Gal-3 mediated by GAGs and proteoglycans. The GAG- and CSPG-binding properties of Gal-3 make the lectin a potential competitor/collaborator of other GAGBPs such as growth factors, cytokines, morphogens, and extracellular matrix proteins.

Drosophila OTK Is a Glycosaminoglycan-Binding Protein with High Conformational Flexibility

SummaryThe transmembrane protein OTK plays an essential role in plexin and Wnt signaling during Drosophila development. We have determined a crystal structure of the last three domains of the OTK ectodomain and found that OTK shows high conformational flexibility resulting from mobility at the interdomain interfaces. We failed to detect direct binding between Drosophila Plexin A (PlexA) and OTK, which was suggested previously. We found that, instead of PlexA, OTK directly binds semaphorin 1a. Our binding analyses further revealed that glycosaminoglycans, heparin and heparan sulfate, are ligands for OTK and thus may play a role in the Sema1a-PlexA axon guidance system.

The Borrelia burgdorferi Glycosaminoglycan Binding Protein Bgp in the B31 Strain Is Not Essential for Infectivity despite Facilitating Adherence and Tissue Colonization

The Lyme disease-causing organism Borrelia burgdorferi is transmitted into the mammalian host by an infected-tick bite. Successful infection relies on the ability of this extracellular pathogen to persist and colonize different tissues. B. burgdorferi encodes a large number of adhesins that are able to interact with host ligands to facilitate adherence and tissue colonization. Multiple glycosaminoglycan binding proteins present in B. burgdorferi offer a degree of redundancy of function during infection, and this highlights the importance of glycosaminoglycans as host cell receptors for spirochete adherence. Of particular interest in this study is Borrelia glycosaminoglycan binding protein (Bgp), which binds to heparin-related glycosaminoglycans. The properties of a bgp transposon mutant and a trans-complemented derivative were compared to those of the wild-type B. burgdorferi in the in vitro binding assays and in infection studies using a C3H/HeJ mouse infection model. We determined that the loss of Bgp impairs spirochete adherence, infectivity, and tissue colonization, resulting in a reduction of inflammatory manifestations of Lyme disease. Although Bgp is not essential for infectivity, it is an important virulence factor of B. burgdorferi that allows adherence and tissue colonization and contributes to disease severity.

Syntheses of chondroitin sulfate tetrasaccharide structures containing 4,6-disulfate patterns and analysis of their interaction with glycosaminoglycan-binding protein.

Chondroitin sulfate tetrasaccharide ligand conjugates, namely GlcA-GalNAc6S-GlcA-GalNAc4S6S (CS-C+E) 1, GlcA2S-GalNAc6S-GlcA2S-GalNAc4S6S (CS-D+T) 2, GlcA-GalNAc4S6S-GlcA-GalNAc4S (CS-E+A) 3, GlcA-GalNAc4S6S-GlcA-GalNAc6S (CS-E+C) 4, and GlcA-GalNAc4S6S-GlcA-GalNAc4S6S (CS-E+E) 5, were systematically synthesized using a disaccharide building block 6. Synthesized CS tetrasaccharide structures were immobilized onto gold-coated chips to prepare array-type sugar chips, and the binding properties of protein were evaluated by surface plasmon resonance imaging biosensor. CS-D+T, CS-E+A, CS-E+C, and CS-E+E showed greater affinity for basic fibroblast growth factor than did other tetrasaccharides (CS-C+D, C+E, D+D).

Immobilization of Heparan Sulfate on Electrospun Meshes to Support Embryonic Stem Cell Culture and Differentiation *

As our understanding of what guides the behavior of multi- and pluripotent stem cells deepens, so too does our ability to utilize certain cues to manipulate their behavior and maximize their therapeutic potential. Engineered, biologically functionalized materials have the capacity to influence stem cell behavior through a powerful combination of biological, mechanical, and topographical cues. Here, we present the development of a novel electrospun scaffold, functionalized with glycosaminoglycans (GAGs) ionically immobilized onto the fiber surface. Bound GAGs retained the ability to interact with GAG-binding molecules and, crucially, presented GAG sulfation motifs fundamental to mediating stem cell behavior. Bound GAG proved to be biologically active, rescuing the neural differentiation capacity of heparan sulfate-deficient mouse embryonic stem cells and functioning in concert with FGF4 to facilitate the formation of extensive neural processes across the scaffold surface. The combination of GAGs with electrospun scaffolds creates a biomaterial with potent applicability for the propagation and effective differentiation of pluripotent stem cells.

Increased heterogeneity of bone matrix mineralization in a patient with familial hypercalcaemic hypocalciuria due to the loss of function of the calcium sensing receptor

Increased heterogeneity of bone matrix mineralization in a patient with familial hypercalcaemic hypocalciuria due to the loss of function of the calcium sensing receptor

Lower bone matrix mineralization in cases of sclerostin deficiency

Lower bone matrix mineralization in cases of sclerostin deficiency

Plasticity of the TSG-6 HA-binding Loop and Mobility in the TSG-6-HA Complex Revealed by NMR and X-ray Crystallography

Tumour necrosis factor-stimulated gene-6 (TSG-6) is a glycosaminoglycan-binding protein expressed during inflammatory and inflammation-like processes. Previously NMR structures were calculated for the Link module of TSG-6 (Link_TSG6) in its free state and when bound to an octasaccharide of hyaluronan (HA 8). Heparin was found to compete for HA binding even though it interacts at a site that is distinct from the HA-binding surface. Here we present crystallography data on the free protein, and 15N NMR relaxation data for the uncomplexed and HA 8-bound forms of Link_TSG6. Although the Link module is comparatively rigid overall, the free protein shows a high degree of mobility in the β4/β5 loop and at the Cys47–Cys68 disulfide bond, both of which are regions involved in HA binding. When bound to HA 8, this dynamic behaviour is dampened, but not eliminated, suggesting a degree of dynamic matching between the protein and sugar that may decrease the entropic penalty of complex formation. A further highly dynamic residue is Lys54, which is distant from the HA-binding site, but was previously shown to be involved in heparin binding. When HA is bound, Lys54 becomes less mobile, providing evidence for an allosteric effect linking the HA and heparin-binding sites. A mechanism is suggested involving the β2-strand and α2-helix. The crystal structure of free Link_TSG6 contains five molecules in the asymmetric unit that are highly similar to the NMR structure and support the dynamic behaviour seen near the HA-binding site: they show little or no electron density for the β4/β5 loop and display multiple conformations for the Cys47–Cys68 disulfide bond. The crystal structures were used in docking calculations with heparin. An extended interface between a Link_TSG6 dimer and heparin 11-mer was identified that is in excellent agreement with previous mutagenesis and calorimetric data, providing the basis for further investigation of this interaction.

Extracellular Secretion of the Borrelia burgdorferi Oms28 Porin and Bgp, a Glycosaminoglycan Binding Protein

Borrelia burgdorferi, the Lyme disease pathogen, cycles between its Ixodes tick vector and vertebrate hosts, adapting to vastly different biochemical environments. Spirochete gene expression as a function of temperature, pH, growth phase, and host milieu is well studied, and recent work suggests that regulatory networks are involved. Here, we examine the release of Borrelia burgdorferi strain B31 proteins into conditioned medium. Spirochetes intrinsically radiolabeled at concentrations ranging from 10(7) to 10(9) cells per ml secreted Oms28, a previously characterized outer membrane porin, into RPMI medium. As determined by immunoblotting, this secretion was not associated with outer membrane blebs or cytoplasmic contamination. A similar profile of secreted proteins was obtained for spirochetes radiolabeled in mixtures of RPMI medium and serum-free Barbour-Stoenner-Kelly (BSK II) medium. Proteomic liquid chromatography-tandem mass spectrometry analysis of tryptic fragments derived from strain B31 culture supernatants confirmed the identity of the 28-kDa species as Oms28 and revealed a 26-kDa protein as 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs-2), previously described as Bgp, a glycosaminoglycan-binding protein. The release of Oms28 into the culture medium is more selective when the spirochetes are in logarithmic phase of growth compared to organisms obtained from stationary phase. As determined by immunoblotting, stationary-phase spirochetes released OspA, OspB, and flagellin. Oms28 secreted by strains B31, HB19, and N40 was also recovered by radioimmunoprecipitation. This is the first report of B. burgdorferi protein secretion into the extracellular environment. The possible roles of Oms28 and Bgp in the host-pathogen interaction are considered.

Dissociation of Heparan Sulfate and Receptor Binding Domains of Hepatocyte Growth Factor Reveals That Heparan Sulfate-c-Met Interaction Facilitates Signaling

Hepatocyte growth factor (HGF) is a secreted, heparan sulfate (HS) glycosaminoglycan-binding protein that stimulates mitogenesis, motogenesis, and morphogenesis in a wide array of cellular targets, including hepatocytes and other epithelial cells, melanocytes, endothelial cells, and hematopoietic cells. NK1 is an alternative HGF isoform that consists of the N-terminal (N) and first kringle (K1) domains of full-length HGF and stimulates all major HGF biological activities. Within NK1, the N domain retains the HS binding properties of full-length HGF and mediates HS-stimulated ligand oligomerization but lacks significant mitogenic or motogenic activity. In contrast, K1 does not bind HS, but it stimulates receptor and mitogen-activated protein kinase activation, mitogenesis, and motogenesis, demonstrating that structurally distinct and dissociable domains of HGF are the primary mediators of HS binding and receptor activation. Despite the absence of HS-K1 binding, K1 mitogenic activity in HS-negative cells is strictly dependent on added soluble heparin, whereas K1-stimulated motility is not. We also found that, like the receptors for fibroblast growth factors, the HGF receptor c-Met binds tightly to HS. These data suggest that HS can facilitate HGF signaling through interaction with c-Met that is independent of HGF-HS interaction and that the recruitment of specific intracellular effectors that mediate distinct HGF responses such as mitogenesis and motility is regulated by HS-c-Met interaction at the cell surface.

A 50 kilodalton protein associated with raf and pp60(v-src) protein kinases is a mammalian homolog of the cell cycle control protein cdc37.

Several oncogenic protein kinases including c-raf-1 and pp60(v-src) are known to directly interact with the 90 kDa heat shock protein (hsp90)/p50 complexes. Using a monoclonal antibody to detect p50 during a purification scheme, p50 was purified to homogeneity. Internal amino acid sequence information was obtained and used to clone a partial cDNA. Comparison of the p50 sequence to other cloned proteins revealed 89% homology with a glycosaminoglycan-binding protein and 54% homology with Drosophila cell cycle control protein (cdc) 37. Monoclonal and polyclonal antibodies were produced against a cleaved fusion protein that recognizes p50 with a high level of specificity. These antibodies recognize the 50 kDa protein present in c-raf-1 and pp60(v-src) complexes. No other proteins were recognized with these antibodies suggesting that p50 is a unique protein. Immunocytochemical visualization of p50 in NIH 3T3 cells indicates a primarily cytoplasmic localization around the nuclear membrane. A survey of p50 expression in murine tissues on a protein blot revealed the following relative levels of expression; thymus > spleen > brain > heart > kidney > liver > lung > skeletal muscle. These results link studies demonstrating complexation of certain kinases with hsp90/p50 in mammalian cells and a number of reports in yeast and Drosophila, demonstrating the importance of cdc37 in cell cycle and kinase function.

Identification and characterization of a divalent cation-dependent glycosaminoglycan-binding protein from rat liver endothelium.

Rat liver endothelial cells (LECs) express a membrane-associated Ca2+-dependent hyaluronan-binding activity (CaHA-BP) which is distinct from the Ca2+-independent, endocytic LEC HA receptor (Yannariello-Brown et al., J. Cell Biochem., 48, 73-80, 1992). The CaHA-BP is specific for a subset of glycosaminoglycans, since Ca2+-dependent binding of 125I-HA (approximately 80 kDa) to LECs was competed with a 100-fold excess (w/w) of HA, chondroitin sulfate, and heparin, but not with chondroitin. The CaHA-BP activity on intact LECs was pH-dependent. Optimal binding occurred at pH 6.0; no binding was detected at pH values <or= 5 or >or= 9. 125I-HA, pre-bound in the presence of Ca2+, could also be dissociated with an acidic buffer (pH 5.0), as well as the divalent cation chelators EDTA and EGTA. 125I-HA binding was stimulated by divalent cations other than Ca2+, such as Mg2+, Mn2+, and Ba2+; with the exception of Zn2+. A photoaffinity crosslinking reagent (125I-ASD-HA) was used to identify specifically crosslinked polypeptides on LECs. In the absence of Ca2+, and in the presence of EGTA, only bands at 175/166 kDa were consistently crosslinked. These bands have been previously identified as the LEC Ca2+-independent endocytic HA receptor (Yannariello-Brown et al., J. Biol. Chem., 267, 20451-20455, 1992). In the presence of Ca2+, crosslinking was consistently seen to a 68 kDa polypeptide. Crosslinking was competed with a 100-fold excess (w/w) of HA. These and other data suggest that a 68 kDa protein is the most likely candidate for the CaHA-BP in LECs.

Characterization of the Glycosaminoglycan-Binding Region of Lactoferrin

Lactoferrin is a prominent component of neutrophil secondary granules and its blood concentration is increased in certain inflammatory diseases. Although the biochemical characterization of lactoferrin as an iron-binding protein has been well described, its physiological role in inflammation remains undefined. We examined the ability of lactoferrin to regulate glycosaminoglycan-accelerated thrombin-serine protease inhibitor (serpin) reactions. Lactoferrin effectively reduced the rate of thrombin-serpin (antithrombin and heparin cofactor II) reactions by three physiological glycosaminoglycans including heparin, heparan sulfate, and dermatan sulfate. An enzyme kinetics analysis showed that lactoferrin did not alter the apparent heparin-thrombin or the heparin-antithrombin dissociation constant values for the heparin-catalyzed thrombin-antithrombin reaction. However, the maximum reaction velocity at saturation with respect to either protein was markedly decreased by lactoferrin. The glycosaminoglycan-binding region of lactoferrin was analyzed following limited proteolysis using Staphylococcus aureus V 8 protease. Two lactoferrin fragments with M r′s of ∼8 and ∼11 kDa were purified based on their affinity to heparin-Sepharose. Amino acid sequence analysis demonstrated that both peptides were from the N-terminus. Although slightly less capable compared to intact lactoferrin, the lactoferrin peptides effectively neutralized heparin, heparan sulfate, and dermatan sulfate-catalyzed serpin-thrombin inhibition reactions. In addition, lactoferrin N-terminal peptides have approximately the same binding affinity to heparin-Sepharose as that of intact lactoferrin. Inspection of both the N-terminal amino acid sequence and the crystal structure of lactoferrin further supports the conclusion that lactoferrin is a novel glycosaminoglycan binding protein and that the putative glycosaminoglycan-binding site is localized to the N-terminus.

Isolation and characterization of a Streptococcus pyogenes protein that binds to basal laminae of human cardiac muscle

A 9-kDa glycosaminoglycan-binding protein (GAG-BP) was isolated from Streptococcus pyogenes and purified to homogeneity by affinity chromatography on heparin-agarose. The protein selectively bound to the basal laminae of human cardiac muscle and had an apparent dissociation constant of 2.5 x 10(-7) M. Chemical analyses indicated that the GAG-BP was rich in alanine, lysine, and arginine (pI 9.5) and devoid of tyrosine, methionine, histidine, and half-cystine. There were no detectable carbohydrate or phosphate substituents. The amino acid sequence of the N terminus of GAG-BP showed homology with those of histone-like DNA-binding proteins of several other bacteria. Circular dichroism spectroscopy indicated that the protein was made up of 50% beta-sheet and 50% beta-turn and random coil in aqueous solution; however, when the protein complexed with heparin, it adopted a more ordered structure containing 25% alpha-helix, 50% beta-sheet, and 25% beta-turn and random coil. The GAG-BP cross-reacted serologically with a component of similar size in extracts of other group A streptococci and was present in the culture medium during late logarithmic growth.

Glycosaminoglycans: molecular properties, protein interactions, and role in physiological processes

Article de synthese sur les glycosaminoglycanes chez les mammiferes: proprietes et structure des proteoglycanes et des proteines de liaison des glycosaminoglycanes; roles dans la regulation des processus cellulaires et la regulation de l'expression des genes; interaction avec les facteurs de transcription