Panels of chemically-modified heparin polysaccharides and natural heparan sulfate saccharides exhibit differences in binding to Slit and Robo, as well as variation between protein binding and cellular activity.

Heparin/ heparan sulfate (HS) glycosaminoglycans are required for Slit-Robo cellular responses. Evidence exists for interactions between each combination of Slit, Robo and heparin/HS and for formation of a ternary complex. Heparin/HS are complex mixtures displaying extensive structural diversity. The relevance of this diversity has been studied to a limited extent using a few select chemically-modified heparins as models of HS diversity. Here we extend these studies by parallel screening of structurally diverse panels of eight chemically-modified heparin polysaccharides and numerous natural HS oligosaccharide chromatographic fractions for binding to both Drosophila Slit and Robo N-terminal domains and for activation of a chick retina axon response to the Slit fragment. Both the polysaccharides and oligosaccharide fractions displayed variability in binding and cellular activity that could not be attributed solely to increasing sulfation, extending evidence for the importance of structural diversity to natural HS as well as model modified heparins. They also displayed differences in their interactions with Slit compared to Robo, with Robo preferring compounds with higher sulfation. Furthermore, the patterns of cellular activity across compounds were different to those for binding to each protein, suggesting that biological outcomes are selectively determined in a subtle manner that does not simply reflect the sum of the separate interactions of heparin/HS with Slit and Robo

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

Crystal structures of the network-forming short-arm tips of the laminin β1 and γ1 chains.

The heterotrimeric laminins are a defining component of basement membranes and essential for tissue formation and function in all animals. The three short arms of the cross-shaped laminin molecule are composed of one chain each and their tips mediate the formation of a polymeric network. The structural basis for laminin polymerisation is unknown. We have determined crystal structures of the short-arm tips of the mouse laminin β1 and γ1 chains, which are grossly similar to the previously determined structure of the corresponding α5 chain region. The short-arm tips consist of a laminin N-terminal (LN) domain that is attached like the head of a flower to a rod-like stem formed by tandem laminin-type epidermal growth factor-like (LE) domains. The LN domain is a β-sandwich with elaborate loop regions that differ between chains. The γ1 LN domain uniquely contains a calcium binding site. The LE domains have little regular structure and are stabilised by cysteines that are disulphide-linked 1-3, 2-4, 5-6 and 7-8 in all chains. The LN surface is not conserved across the α, β and γ chains, but within each chain subfamily there is a striking concentration of conserved residues on one face of the β-sandwich, while the opposite face invariably is shielded by glycans. We propose that the extensive conserved patches on the β and γ LN domains mediate the binding of these two chains to each other, and that the α chain LN domain subsequently binds to the composite β-γ surface. Mutations in the laminin β2 LN domain causing Pierson syndrome are likely to impair the folding of the β2 chain or its ability to form network interactions

Determinants of laminin polymerization revealed by the structure of the alpha 5 chain amino-terminal region

The polymerization of laminin into a cell-associated network—a key step in basement membrane assembly—is mediated by the laminin amino-terminal (LN) domains at the tips of the three short arms of the laminin αβγ-heterotrimer. The crystal structure of a laminin α5LN–LE1–2 fragment shows that the LN domain is a β-jelly roll with several elaborate insertions that is attached like a flower head to the stalk-like laminin-type epidermal growth factor-like tandem. A surface loop that is strictly conserved in the LN domains of all α-short arms is required for stable ternary association with the β- and γ-short arms in the laminin network

Location of Pierson syndrome mutations.

<p>Shown is a cartoon drawing of the first two domains of the laminin β1 LN-LEa1-4 structure (LN, blue; LEa1, green; disulphide bridges, yellow; <i>N</i>-linked glycan; dark pink), in which residues corresponding to those mutated in the laminin β2 chain of Pierson syndrome patients <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042473#pone.0042473-Matejas1" target="_blank">[15]</a> are shown in red. Only homozygous missense mutations are shown. The view direction is similar to the right panel of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042473#pone-0042473-g002" target="_blank">Figure 2A</a>.</p

Schematic drawing of the laminin-511 heterotrimer.

<p>The α5 chain is shown in grey. In the β1 and γ1 chain, the regions corresponding to the crystal structures described in this report are coloured. LN, laminin N-terminal domain; LE, laminin-type epidermal growth factor-like domain.</p

Crystallographic statistics.

a<p>Values in parantheses are for the highest resolution shell.</p>b<p>Difference in B-factors of atoms connected by a covalent bond.</p>c<p>Residues in core, allowed, generously allowed and disallowed regions of the Ramachandran plot <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042473#pone.0042473-Laskowski1" target="_blank">[44]</a>.</p

Pairwise sequence identities between LN-LEa1-2 regions of mouse laminin chains.

<p>Pairwise sequence identities between LN-LEa1-2 regions of mouse laminin chains.</p

Simulated annealing omit maps of selected model regions.

<p>Partial models, from which regions of interest were omitted, were subjected to simulated annealing refinement from 3000 K to remove model bias. Shown are SIGMAA-weighted 2F_obs-F_calc maps calculated from the partial models after refinement. (A) Stereoview of LEa1 in the laminin β1 LN-LEa1–4 structure. Residues 270–310 were omitted from the map calculation (R_free = 0.333). The disulphide bridges are labelled with the sequence numbers of the linked cysteines. According to the mass spectrometric analysis of Kalkhof et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042473#pone.0042473-Kalkhof2" target="_blank">[20]</a>, Cys271 is linked to Cys273, Cys280 unpaired, Cys298 linked to Cys300, and Cys309 linked to Cys313 (not visible). (B) Stereoview of the calcium binding loop in the laminin γ1 LN-LEa1–2 structure. Residues 102–115 and the calcium ion were omitted from refinement (R_free = 0.315). The green electron density is a F_obs-F_calc difference density map contoured at 7σ, confirming the presence of a heavy atom at the position of the calcium ion.</p