Synthesis of heterotrimeric collagen peptides containing the α1β1 integrin recognition site of collagen type IV

Abstract

Collagen type IV provides a biomechanically stable scaffold into which the other constituents of basement membranes are incorporated, but it also plays an important role in cell adhesion. This occurs with collagen type IV mainly via the alpha1beta1 integrin, and the proposed epitope involved in this type of collagen/integrin interaction corresponds to a non-sequential R/Xaa/D motif, where the arginine and aspartate residues are provided by the alpha2 and alpha1 chains of the collagen molecule, respectively. Since the stagger of the three alpha chains in native collagen type IV is still unknown and different alignments of the chains lead to different spatial epitopes, two heterotrimeric collagen peptides containing the natural 457-469 sequences of the cell adhesion site were synthesized in which the single chains were assembled via disulfide bonds into the two most plausible alpha1alpha2alpha1' and alpha2alpha1alpha1' registers. The differentiated triple-helical stabilities of the two heterotrimers suggest a significant structural role of the chain register in collagen, although the binding to alpha1beta1 integrin is apparently less affected as indicated by preliminary experiments.

Synthetic heterotrimeric collagen peptides as mimics of cell adhesion sites of the basement membrane

“…Conversely, for the correct registration and assembly of heterotrimeric collagen peptides containing functional sequence portions of natural collagens, we have introduced a simple artificial C-terminal cystine knot exploiting regioselective cysteine pairing procedures to align the three peptide chains via two interchain-disulfide bridges into the desired staggers. 29,31,[53][54][55] Modeling and molecular dynamics (MD) simulations served to optimally fit the cystine knot into the triple-helix without steric clashes, 29 a fact that was recently confirmed by NMR structural analysis of a model peptide containing such disulfide connectivities. 56 In view of the positive experiences with these disulfide-cross-linked collagenous heterotrimers and also because of the presence of a cystine knot downstream from the ␣1␤1 adhesion epitope, the two heterotrimers A and B ( Figure 2) were selected as possible mimics of the ␣1␤1 adhesion epitope of collagen type IV.…”

“…56 In view of the positive experiences with these disulfide-cross-linked collagenous heterotrimers and also because of the presence of a cystine knot downstream from the ␣1␤1 adhesion epitope, the two heterotrimers A and B ( Figure 2) were selected as possible mimics of the ␣1␤1 adhesion epitope of collagen type IV. 55 These trimers contain the sequence portions 457-468 of the aligned ␣1 and ␣2 chains of collagen type IV which encompass the residues 461 directly involved in binding to ␣1␤1 integrin. While in the trimer B the synthetic peptide chains are aligned in the ␣2␣1␣1Ј register with ␣2 as the leading chain according to the stagger proposed for collagen type IV, 15 in the trimer A the less probable ␣1␣2␣1Ј register was applied to analyze the effect of the chain register on integrin binding affinities.…”

“…53,54 By applying this strategy to the assembly of the trimers A and B difficulties were encountered because of the high tendency, particularly, of the ␣1 chain to self-associate into homotrimers. 55 Therefore, independently of the target trimer, thiol activation was performed only on the ␣2 chain as shown in Scheme 1. Moreover, activation of the intermediate dimers by displacement of the S-Acm derivative with Npys-Cl for final assembly of the trimers had to be performed under kinetic control to avoid extensive scrambling of the preformed dimer.…”

Methods for Identifying Novel Integrin Ligands

“…However, not all integrins recognize the RGD sequence, and some integrins recognize sites consisting of amino acids from more than one protein subunit, such as sequences in the triple helical domain of collagen (Sacca and Moroder, 2002). In addition to overlooking ligands, several of the abovementioned assays may identify ligands that are not relevant at physiological concentrations of integrin and ligand.…”

“…Another approach, peptide display by filamentous phage, has been used successfully to identify peptide ligands, such as integrin-ligand mimetics (Koivunen et al, 1994(Koivunen et al, , 1995, but has not identified physiologically relevant ligands. However, this may not be an ideal approach since the binding site of ligands is often derived from multiple segments of the protein that may not be contiguous (Sacca and Moroder, 2002).…”