Computational modeling of laminin N-terminal domains using sparse distance constraints from disulfide bonds and chemical cross-linking.

Basement membranes are thin extracellular protein layers, which separate endothelial and epithelial cells from the underlying connecting tissue. The main noncollagenous components of basement membranes are laminins, trimeric glycoproteins, which form polymeric networks by interactions of their N-terminal (LN) domains; however, no high-resolution structure of laminin LN domains exists so far. To construct models for laminin ß(1) and γ(1) LN domains, 14 potentially suited template structures were determined using fold recognition methods. For each target/template-combination comparative models were created with Rosetta. Final models were selected based on their agreement with experimentally obtained distance constraints from natural cross-links, that is, disulfide bonds as well as chemical cross-links obtained from reactions with two amine-reactive cross-linkers. We predict that laminin ß(1) and γ(1) LN domains share the galactose-binding domain-like fold.

A novel disulfide pattern in laminin-type epidermal growth factor-like (LE) modules of laminin ß1 and γ1 chains.

In-depth mass spectrometric analysis of disulfide bond patterns in recombinant mouse laminin ß1 and γ1 chain N-terminal fragments comprising the laminin N-terminal (LN) domain and the first four laminin epidermal growth factor-like (LE) domains revealed a novel disulfide pattern for LE domains. This showed a (2-3, 4-5, 6-7, 8-1) connectivity with the last cysteine of one LE domain being connected to the first cysteine of the following LE domain. The same pattern was also found in E4, the N-terminal ß1 chain fragment derived by elastase digestion of mouse EHS tumor laminin-111, showing that this pattern occurs in native laminin. The strictly linear pattern with an interdomain disulfide has not been described previously for EGF domains. The N-terminal portions of laminin short arms, consisting of the LN domain and LE domains 1-4, are essential for laminin-laminin self-interactions, whereas the internal LE domains 7-9 in the laminin γ1 chain harbor the nidogen binding site and have a conventional disulfide pattern. This suggests that LE domains differing in function also differ in their disulfide patterns.

Determination of disulfide bond patterns in laminin beta1 chain N-terminal domains by nano-high-performance liquid chromatography/matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry.

The disulfide bonding patterns in the N-terminal (LN) domains of the basement membrane protein laminin beta1 have not been investigated so far. We report an in-depth mass spectrometric analysis using offline nano-high-performance liquid chromatography/matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (nano-HPLC/MALDI-TOF/TOF-MS) for determining the disulfide bond patterns in the LN-domain of recombinant mouse laminin beta1 chain for the first time. Mass spectra were recorded and the putatively disulfide-linked peptides were subjected to LIFT-TOF/TOF-MS to confirm the disulfide bond. Screening the fragment ion mass spectra of disulfide-linked peptides for characteristic 66-amu patterns (34 u +32 u), arising from symmetric and asymmetric cleavage of disulfide bonds, facilitated their identification. Using various enzymes for proteolytic digestion of a recombinant laminin beta1 chain N-terminal protein fragment, a linear bonding pattern of the eight cysteine residues in the LN-domain of the laminin beta1 chain was observed with a (1-2, 3-4, 5-6, 7-8) connectivity of cysteines. The identical disulfide-bonding pattern was found in E4, the N-terminal laminin beta1 chain fragment derived by elastase digestion of mouse tumor laminin-111, confirming that this pattern also occurs in native laminin.

Molecular analysis of laminin N-terminal domains mediating self-interactions.

The ability of laminins to self-polymerize is crucial for the formation of basement membranes. Development of this polymerized network has profound effects upon tissue architecture as well as on the intracellular organization and differentiation of neighboring cells. The laminin N-terminal (LN) domains have been shown to mediate this interaction and studies using proteolytic fragments derived from laminin-1 led to the theory that network assembly depends on the formation of a heterotrimeric complex between LN domains derived from alpha, beta, and gamma chains in different laminin molecules with homologous interactions being insignificant. The laminin family consists of 15 known isoforms formed from five alpha, three beta, and three gamma chains, of which some are truncated and lack the N-terminal LN domain. To address whether the model of heterotrimeric complex formation is applicable to laminin isoforms other than laminin-1, eight LN domains found in the laminin protein family were recombinantly expressed and tested in three different assays for homologous and heterologous interactions. The results showed that the lack of homologous interactions is an exception, with such interactions being seen for LN domains derived from all alpha chains and from the beta2 and beta3 subunits. The gamma chain-derived LN domains showed a far more limited binding repertoire, particularly in the case of the gamma3 chain, which is found present in a range of non-basement membrane locations. Further, whereas the interactions depended upon Ca2+ ions, with EDTA reversibly abrogating binding, EDTA-induced conformational changes were not reversible. Together these results demonstrate that the assembly model proposed on the basis of laminin-1 may be a simplification, with the assembly of naturally occurring laminin networks being far more complex and highly dependent upon which laminin isoforms are present.

Signals from load sensors underlie interjoint coordination during stepping movements of the stick insect leg.

During stance and swing phase of a walking stick insect, the retractor coxae (RetCx) and protractor coxae (ProCx) motoneurons and muscles supplying the thorax-coxa (TC)-joint generate backward and forward movements of the leg. Their activity is tightly coupled to the movement of the more distal leg segments, i.e., femur, tibia, and tarsus. We used the single middle leg preparation to study how this coupling is generated. With only the distal leg segments of the middle leg being free to move, motoneuronal activity of the de-afferented and -efferented TC-joint is similarly coupled to leg stepping. RetCx motoneurons are active during stance and ProCx motoneurons during swing. We studied whether sensory signals are involved in this coordination of TC-joint motoneuronal activity. Ablation of the load measuring campaniform sensilla (CS) revealed that they substantially contribute to the coupling of TC-joint motoneuronal activity to leg stepping. Individually ablating trochanteral and femoral CS revealed the trochanteral CS to be necessary for establishing the coupling between leg stepping and coxal motoneuron activity. When the locomotor system was active and generated alternating bursts of activity in ProCx and RetCx motoneurons, stimulation of the CS by rearward bending of the femur in otherwise de-afferented mesothoracic ganglion terminated ongoing ProCx motoneuronal activity and initiated RetCx motoneuronal activity. We show that cuticular strain signals from the trochanteral CS play a major role in shaping TC-joint motoneuronal activity during walking and contribute to their coordination with the stepping pattern of the distal leg joints. We present a model for the sensory control of timing of motoneuronal activity in walking movements of the single middle leg.