Rational designation and characterization of a novel humanized collagen capable of self-assembling into triple helix and fibrils with D-period

The triple helix and D-period are distinctive features of native collagen, crucial for its physicochemical properties and bioactivities. However, developing recombinant humanized collagen with D-period features remains elusive. Here, we present a strategy for preparing a novel recombinant humanized collagen using a ‘charged-hydrophobic-charged amino acid’ sequence with the capacity of self-assembling. The hydrophobic amino acids in the middle region are believed to be crucial for the triple helix formation while the charged amino acids at the C- and N-terminal drive the triple-helix to self-assemble into higher-order structures like fibrils, with D-period formation during this process. To prove this concept, the particular fragment of Gly1059-Ala1103 of human type III collagen, featuring arginine (R), lysine (K), aspartic acid (D), and glutamic acid (E)-rich termini and a Glycine-Proline-Alanine (G-P-A) central motif, was selected and repeated to construct a recombinant humanized collagen, designated as rhCL04. This construct successfully formed hierarchical structures, including triple helices, rod-like fibrils, and hydrogels, exhibiting a distinct 10 nm D-period across a broad pH range from 4 to 10. Additionally, cell adhesion and biocompatibility were confirmed using L929 mouse fibroblast cells, demonstrating the ability to promote cell adhesion activity and no significant cytotoxicity. Our study provides valuable insights into the self-assembling mechanisms of native collagens. Moreover, these results highlight the efficacy of this strategy in producing recombinant humanized collagen with collagen-like characteristics. The simplicity and versatility of the approach, combined with the excellent self-assembling properties and biological activity of rhCL04, underscore its potential for biomaterial production.