ABSTRACT

Self‐assembling peptides have attracted increasing attention as building blocks for developing functional materials. Among them, collagen‐mimetic peptides (CMPs) represent a class of self‐assembling systems. However, although short‐chain CMPs spontaneously fold into thermodynamically stable triple‐helical trimers, they fail to further undergo supramolecular assembly to form hydrogels. We report a new strategy for supramolecular hydrogel formation based on CMPs. The designed peptide, termed kinkCMP, features a kinked backbone introduced at the center of the sequence via adjacent disulfide bonds. The kinkCMP forms a gel, and its gel denaturation temperature can be tuned by varying the peptide chain length. Moreover, the hydrogel can be used as a cell culture scaffold. These results provide a versatile approach for constructing supramolecular hydrogels based on CMPs, expanding the design strategies for peptide‐based functional materials.

Integrins α1β1, α2β1, α10β1, and α11β1 serve as primary collagen receptors, recognizing the GxxGEx motif of the collagen triple helix via their αI domains. The Glu residue of the motif coordinates with a divalent metal cation at the metal ion-dependent adhesion site of the αI domain. Owing to this conserved recognition mechanism, previously identified binding sequences show low subtype selectivity. In this study, we identified triple-helical peptide ligands capable of selectively binding to the α2I domain in a manner independent of both Glu and a divalent metal cation. By employing yeast-two hybrid screening using a randomized triple-helical peptide library, we identified sequences in which canonical Glu was substituted by aliphatic residues such as Met. X-ray crystallographic analysis of the complex formed by the α2I domain and a triple-helical peptide containing GFOGMR (O: 4-hydroxyproline)—a Met-substituted variant of the canonical integrin-binding sequence GFOGER—revealed a novel binding mode. In this mode, the peptide was recognized by the closed form of the α2I domain in a metal cation-independent manner. Furthermore, this interaction mode enabled Met-containing peptides to specifically bind to the integrin α2I domain. These findings deepen our understanding of collagen recognition by collagen-binding integrins and provide insight for the discovery of ligands capable of selectively targeting collagen receptor-type integrins.

ABSTRACT

The fibrous structures of collagen provide physical strength and stability to tissues and organs. Abnormalities in their orientation, growth, and remodeling cause morphogenetic defects and serious diseases including fibrosis, so it is important to clarify how collagen fibers are correctly oriented and grown within tissues. However, this mechanism remains elusive, as few methods have been available to fluorescently stain collagen fibers with a simple protocol and to observe their structure in three dimensions. Here we present a facile method that enables fluorescent staining of collagen fibers in vertebrate tissues. In our method using DAF-FM, known as a NO detection probe, premature collagen fibers can be visualized via covalent binding to the allysine residues serving as precursors of cross-linking structures of collagen. In addition, we showed that the labeling method using two fluorescent probes with different colors, DAF-FM and DAR-4M, allows for pulse-chase observation of newly synthesized collagen fibers. Our method will be a breakthrough technique in future collagen studies.

Triple helix formation of procollagen occurs in the endoplasmic reticulum (ER) where the single-stranded α-chains of procollagen undergo extensive post-translational modifications. The modifications include prolyl 4- and 3-hydroxylations, lysyl hydroxylation, and following glycosylations. The modifications, especially prolyl 4-hydroxylation, enhance the thermal stability of the procollagen triple helix. Procollagen molecules are transported to the Golgi and secreted from the cell, after the triple helix is formed in the ER. In this study, we investigated the relationship between the thermal stability of the collagen triple helix and environmental temperature. We analyzed the number of collagen post-translational modifications and thermal melting temperature and α-chain composition of secreted type I collagen in zebrafish embryonic fibroblasts (ZF4) cultured at various temperatures (18, 23, 28, and 33 °C). The results revealed that thermal stability and other properties of collagen were almost constant when ZF4 cells were cultured below 28 °C. By contrast, at a higher temperature (33 °C), an increase in the number of post-translational modifications and a change in α-chain composition of type I collagen were observed; hence, the collagen acquired higher thermal stability. The results indicate that the thermal stability of collagen could be autonomously tuned according to the environmental temperature in poikilotherms.

Heat shock protein 47 (HSP47) is an endoplasmic reticulum (ER)-resident molecular chaperone that specifically recognizes triple helical portions of procollagens. The chaperone function of HSP47 is indispensable in mammals, and hsp47-null mice show an embryonic lethal phenotype accompanied by severe abnormalities in collagen-based tissue structures. Two leading hypotheses are currently accepted for the molecular function of HSP47 as a procollagen-specific chaperone. One is facilitation of procollagen folding by stabilizing thermally unstable triple helical folding intermediates, and the other is inhibition of procollagen aggregation or lateral association in the ER. The aim of this study was to elucidate the functional essence of this unique chaperone using fibroblasts established from hsp47-/- mouse embryos. When the cells were cultured at 37 °C, various defects in procollagen biosynthesis were observed, such as accumulation in the ER, over-modifications including prolyl hydroxylation, lysyl hydroxylation, and further glycosylation, and unusual secretion of type I collagen homotrimer. All defects were corrected by culturing the cells at a lower temperature of 33 °C. These results indicated that lowering the culture temperature compensated for the loss of HSP47. This study elucidated that HSP47 stabilizes the elongating triple helix of procollagens, which is otherwise unstable at the body temperature of mammals.