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Jeremy E Turnbull

Publications and source records attributed to Jeremy E Turnbull.

16 recordsLinked to original sources
PubMed2006

A molecular mechanism for the heparan sulfate dependence of slit-robo signaling.

Slit is a large secreted protein that provides important guidance cues in the developing nervous system and in other organs. Signaling by Slit requires two receptors, Robo transmembrane proteins and heparan sulfate (HS) proteoglycans. How HS controls Slit-Robo signaling is unclear. Here we show that the second leucine-rich repeat domain (D2) of Slit, which mediates binding to Robo receptors, also contains a functionally important binding site for heparin, a highly sulfated variant of HS. Heparin markedly enhances the affinity of the Slit-Robo interaction in a solid-phase binding assay. Analytical gel filtration chromatography demonstrates that Slit D2 associates with a soluble Robo fragment and a heparin-derived oligosaccharide to form a ternary complex. Retinal growth cone collapse triggered by Slit D2 requires cell surface HS or exogenously added heparin. Mutation of conserved basic residues in the C-terminal cap region of Slit D2 reduces heparin binding and abolishes biological activity. We conclude that heparin/HS is an integral component of the minimal Slit-Robo signaling complex and serves to stabilize the relatively weak Slit-Robo interaction.

Amino Acid Sequence↗
PubMed2006

Heparin derivatives as inhibitors of BACE-1, the Alzheimer's beta-secretase, with reduced activity against factor Xa and other proteases.

Heparan sulfate (HS) regulates processing of the amyloid precursor protein by the Alzheimer's beta-secretase (BACE-1). An HS analogue, porcine intestinal mucosal heparin, was systematically modified at the principal positions of O-sulfation and N-sulfation/acetylation and tested for BACE-1 inhibitory and anti factor Xa activities. The derivative with the highest anti-BACE-1 to anti-Xa activity ratio contained N-acetyl and 2-O- and 6-O-sulfates and also exhibited attenuated activities against cathepsin-D and renin, two other structurally related aspartyl proteases.

Alzheimer Disease↗
PubMed2006

Engineered bio-active polysaccharides from heparin.

[Image: see text] Heparin, the well-known anticoagulant polysaccharide, is also active in many other biological systems owing to its structural similarity to HS, but usually lacks selectivity because it is more highly sulfated. A series of straightforward chemical reactions (de-O-sulfation, de-N-sulfation and re-N-acetylation), carried out to partial or complete extent, were combined, resulting in a number of modified heparin polysaccharide derivatives with altered properties. These exhibited a range of abilities to promote cell signalling through the FGF/FGFR tyrosine kinase signalling system, in an in vitro cell assay with combinations of FGF-1, -2, -3 and FGFR 1 and 3. One polysaccharide (N-acetylated, 6-O- and 2-O-sulfated heparin), with only a fraction (<10(-3)) of the anticoagulant activity of heparin (200 U . mg(-1)), promoted FGF-2-mediated angiogenesis (10-fold) and therefore had an improved ratio of pro-angiogenic activity to anticoagulant activity in excess of 10(4) compared to heparin. These results demonstrate that heparin-derived polysaccharides can be engineered for selected activities and have potential in a wide range of medical, biotechnological and tissue-engineering applications. Effect of selected engineered heparin polysaccharides on angiogenesis.

Anticoagulants↗
PubMed2006

Fabrication of carbohydrate microarrays on gold surfaces: direct attachment of nonderivatized oligosaccharides to hydrazide-modified self-assembled monolayers.

This paper describes a new and simple microarray platform for presenting multiple nonderivatized oligosaccharides to protein targets, with utility for mapping carbohydrate-protein recognition events. The approach is based on the creation of a hydrazide-derivatized, self-assembled monolayer on a gold surface in a single or two-step procedure, for efficient and selectively oriented anchoring of oligosaccharide probes via their reducing ends, with detection using fluorescence detection of bound proteins. The biggest hurdles in employing gold-based substrate for fluorescence-based microarray detection include fluorescence quenching and nonspecific surface adsorption of proteins. We found that the quenching effect could be minimized by introducing a omega-thiolated fatty acid (C16) self-assembled monolayer between the gold surface and hydrazide groups, followed by detection involving three successive binding protein layers covering the gold surface. In addition, an effective blocking scheme involving poly(ethylene glycol) aldehyde and bovine serum albumin was employed to reduce nonspecific protein adsorption to the chip surface. As proof of principle, we demonstrate here that sulfated oligosaccharide probes from heparin can be effectively and covalently attached without prior derivatization onto the hydrazide-modified, self-assembled monolayer on gold-coated slide surfaces in a microarray format. This platform is used to assess binding of specific heparin-binding protein targets at very high sensitivity, and we also demonstrate that the approach can be extended to nonsulfated sugars. Direct attachment of nonderivatized sugar probes on the chip is advantageous since it avoids the need for laborious prederivatization and cleanup steps. This versatile fluorescence microarray platform provides a facile approach for interrogating multiple carbohydrate-protein interactions in a high-throughput manner and has potential as a common gold surface platform for other diverse interrogations by MALDI-MS, surface plasmon resonance, and quartz crystal microbalances.

Gold↗
PubMed2005

Interactions of multiple heparin binding growth factors with neuropilin-1 and potentiation of the activity of fibroblast growth factor-2.

The hypothesis that neuropilin-1 (Npn-1) may interact with heparin-binding proteins other than vascular endothelial growth factor has been tested using an optical biosensor-based binding assay. The results show that fibroblast growth factor (FGF) 1, 2, 4, and 7, FGF receptor 1, hepatocyte growth factor/scatter factor (HGF/SF), FGF-binding protein, normal protease sensitive form of prion protein, antithrombin III, and Npn-1 itself are all able to interact with Npn-1 immobilized on the sensor surface. FGF-2, FGF-4, and HGF/SF are also shown to interact with Npn-1 in a solution assay. Moreover, these protein-protein interactions are dependent on the ionic strength of the medium and are inhibited by heparin, and the kinetics of binding of FGF-2, FGF-4 and HGF/SF to Npn-1 are characterized by fast association rate constants (270,000-1,600,000 m(-1) s(-1)). These results suggest that Npn-1 possesses a "heparin" mimetic site that is able to interact at least in part through ionic bonding with the heparin binding site on many of the proteins studied. Npn-1 was also found to potentiate the growth stimulatory activity of FGF-2 on human umbilical vein endothelial cells, indicating that Npn-1 may not just bind but also regulate the activity of heparin-binding proteins.

Animals↗
PubMed2005

Heparan 2-O-sulfotransferase, hst-2, is essential for normal cell migration in Caenorhabditis elegans.

The importance of heparan sulfate proteoglycans has been highlighted by a number of human genetic disorders associated with mutations in genes encoding for heparan sulfate proteoglycan protein cores or biosynthetic enzymes required for heparan sulfate (HS) assembly. To study the functional role of HS in Caenorhabditis elegans development cosmid sequence C34F6.4 was identified as the C. elegans ortholog of vertebrate heparan 2-O-sulfotransferase (HS2ST) and the gene named hst-2. HS2ST activity is present in C. elegans and is completely absent in a deletion mutant of hst-2, ok595, and specifically reduced by hst-2 RNA interference. Expression of hst-2 in CHO cells deficient in HS2ST rescues enzyme activity and binding of FGF2 to cell surface HS. hst-2 expression is found in the hypodermis, muscle, distal tip cells (DTCs), and in neurons. A null mutation in hst-2 causes cell migration defects. This work demonstrates sulfotransferase activity in C. elegans and indicates that specific 2-O-sulfate modifications are critical for normal HS functions in controlling cell migration.

Animals↗
PubMed2004

Proteoglycans make the grade-ient.

Two recent reports in Developmental Cell reveal further insights into the complex mechanisms by which heparan sulfate proteoglycans provide organisms with novel ways of generating and interpreting morphogen gradients.

Animals↗
PubMed2004

Interactions of heparin/heparan sulfate with proteins: appraisal of structural factors and experimental approaches.

Over the past decade, the glycosaminoglycans heparin and heparan sulfate have been shown to bind and regulate the activities of many proteins. Established techniques have provided both qualitative and quantitative information regarding these interactions, leading to a general view that proteins bind with a variety of affinities to particular sequences within heparin or heparan sulfate chains. The mechanism by which heparan sulfate regulates the activity of proteins through such interactions has, however, proved more elusive. We survey some relevant details of the structural characteristics of heparin/heparan sulfate and the approaches used to investigate their interactions with proteins. For the latter, the interactions of heparin/heparan sulfate with fibroblast growth factors and their receptors will be emphasized, because these proteins have been the subject of many studies. We reflect on the information that various techniques have provided, points regarding their use, and some relevant theoretical considerations regarding the study of protein-heparin/heparan sulfate interactions. A perspective of new and developing approaches, which may aid advances in this field, is also provided.

Carbohydrate Conformation↗
PubMed2004

Highly diverse heparan sulfate analogue libraries: providing access to expanded areas of sequence space for bioactivity screening.

Structurally diverse heparan sulfate analogue libraries were produced chemicoenzymatically from heparin. They possess vastly more heterogeneity than tissue heparan sulfates, expand the sequence space available for screening, and can help identify minimal structural features associated with activity. Library components are likely to exhibit fewer nonspecific interactions and side-effects than heparin or simple chemically modified heparin. A strategy for their use is illustrated for the fibroblast growth factor-receptor tyrosine kinase signaling system.

Cell Line↗
PubMed2003

Heparan sulfate regulates amyloid precursor protein processing by BACE1, the Alzheimer's beta-secretase.

Cleavage of amyloid precursor protein (APP) by the Alzheimer's beta-secretase (BACE1) is a key step in generating amyloid beta-peptide, the main component of amyloid plaques. Here we report evidence that heparan sulfate (HS) interacts with beta-site APP-cleaving enzyme (BACE) 1 and regulates its cleavage of APP. We show that HS and heparin interact directly with BACE1 and inhibit in vitro processing of peptide and APP substrates. Inhibitory activity is dependent on saccharide size and specific structural characteristics, and the mechanism of action involves blocking access of substrate to the active site. In cellular assays, HS specifically inhibits BACE1 cleavage of APP but not alternative cleavage by alpha-secretase. Endogenous HS immunoprecipitates with BACE1 and colocalizes with BACE1 in the Golgi complex and at the cell surface, two of its putative sites of action. Furthermore, inhibition of cellular HS synthesis results in enhanced BACE1 activity. Our findings identify HS as a natural regulator of BACE1 and suggest a novel mechanism for control of APP processing.

Alzheimer Disease↗
PubMed2002

Fibroblast growth factor receptors 1 and 2 interact differently with heparin/heparan sulfate. Implications for dynamic assembly of a ternary signaling complex.

Heparan sulfate (HS) regulates the kinetics of fibroblast growth factor 2 (FGF2)-stimulated intracellular signaling and differentially activates cell proliferation of cells expressing different FGF receptors (FGFRs). Evidence suggests that HS interacts with both FGFs and FGFRs to form active ternary signaling complexes. Here we compare the interactions of two FGFRs with HS. We show that the ectodomains of FGFR1 IIIc and FGFR2 IIIc exhibit specific interactions with different characteristics for both heparin and porcine mucosal HS. These glycans are both known to activate FGF signaling via these receptors. FGFR2 interacts with a higher apparent affinity than FGFR1 despite both involving 6-O-, 2-O-, and N-sulfates. FGFR1 and FGFR2 bind heparin with mean association rate constants of 1.9 x 10(5) and 2.1 x 10(6) m(-1)s(-1), respectively, and dissociation rate constants of 1.2 x 10(-2) and 2.7 x 10(-2) s(-1), respectively. These produced calculated affinities of 63 and 13 nm, respectively. Hence, FGFR1 and FGFR2 bind to heparin chains with markedly different kinetics and affinities. We propose a mechanistic model where the kinetic parameters of the HS/FGFR interaction are a key element regulating the formation of ternary complexes and the resulting FGF signaling outcomes.

Animals↗
PubMed2002

Identification of the heparan sulfate binding sites in the cellular prion protein.

Data from cell culture and animal models of prion disease support the separate involvement of both heparan sulfate proteoglycans and copper (II) ions in prion (PrP) metabolism. Though direct interactions between prion protein and heparin have been recorded, little is known of the structural features implicit in this interaction or of the involvement of copper (II) ions. Using biosensor and enzyme-linked immunosorbent assay methodology we report direct heparin and heparan sulfate-binding activity in recombinant cellular prion protein (PrP(c)). We also demonstrate that the interaction of recombinant PrP(c) with heparin is weakened in the presence of Cu(II) ions and is particularly sensitive to competition with dextran sulfate. Competitive inhibition experiments with chemically modified heparins also indicate that 2-O-sulfate groups (but not 6-O-sulfate groups) are essential for heparin recognition. We have also identified three regions of the prion protein capable of independent binding to heparin and heparan sulfate: residues 23-52, 53-93, and 110-128. Interestingly, the interaction of an octapeptide-spanning peptide motif amino acids 53-93 with heparin is enhanced by Cu(II) ions. Significantly, a peptide of this sequence is able to inhibit the binding of full-length prion molecule to heparin, suggesting a direct role in heparin recognition within the intact protein. The collective data suggest a complex interaction between prion protein and heparin/heparan sulfate and has implications for the cellular and pathological functions of prion proteins.

Animals↗
PubMed2002

Variant heparan sulfates synthesized in developing mouse brain differentially regulate FGF signaling.

Heparan sulfates (HSs) exert critical regulatory actions on many proteins, including growth factors, and are essential for normal development. Variations in their specific sulfation patterns are known to regulate binding and signaling of fibroblast growth factors (FGFs) via tyrosine kinase receptors (FGFRs). We previously reported differences in sulfation patterns between HS species expressed by embryonic day 10 (E10) and E12 mouse neural precursor cells. We have examined the abilities of the different HS species to support signaling of the relevant FGF-FGFR combinations expressed early during brain development. For FGF8, which only functions early (E8-E11), E10 HS showed preferential activation. The most potent signaling for FGF8 was via FGFR3c, for which E10 HS was strongly active and E12 HS had no activity. For FGF2, which functions from E10 to E13, HS from both stages showed similar activity and were more potent at activating FGFR1c than the other receptors. Thus, we find a stage-specific correlation with activation. To explore the potential mechanisms for the generation of these stage-specific HS species, we investigated the expression of the HS sulfotransferase (HSST) isozymes responsible for creating diverse sulfation motifs in HS chains. We find that there are stage-specific combinations of HSST isozymes that could underlie the synthesis of different HS species at E10 and E12. Collectively, these data lead us to propose a model in which differential expression of HSSTs results in the synthesis of variant HS species that form functional signaling complexes with FGFs and FGFRs and orchestrate proliferation and differentiation in the developing brain.

Alternative Splicing↗
PubMed2002

Specific heparan sulfate structures involved in retinal axon targeting.

Heparan sulfate (HS), a structurally diverse molecule comprising distinct sequences of sulfated disaccharide units, is abundant in the developing brain and binds to axon guidance molecules. Addition of HS to the developing Xenopus optic pathway causes severe targeting errors yet it is not known how the structural diversity of this molecule relates to its role in axon guidance. We have used an in vivo brain assay to identify the structural characteristics of HS that induce aberrant axon targeting. Inhibiting sulfation of endogenous HS with chlorate causes axons to bypass their target, the tectum, and treatment with chemically modified heparins reveals that 2-O- and 6-O-sulfate groups have potent bypass-inducing activity. Experiments with purified heparin saccharides show that bypass-inducing activity correlates with distinct structures, particularly those containing a combination of 2-O- and 6-O-sulfate groups. Taken together the results indicate that specific sequences, rather than gross structural composition, are critical for activity. In situ hybridisation revealed that HS 6-O-sulfotransferase is regionally expressed along the border of the dorsal optic tract whereas 2-O-sulfotransferase is expressed broadly. Our results demonstrate that specific HS sequences are essential for regulating retinotectal axon targeting and suggest that regionalised biosynthesis of specific HS structures is important for guiding axons into the tectum.

Animals↗