Progress in the detection and quantification of collagens: a review

Collagens are an important and ubiquitous family of proteins. They have many functions in the human body and similarly have found numerous, potent applications in various industries including the manufacture of biomaterials. The ever-increasing demand for collagen has made necessary the exploration of alternative sources such as bacterial collagen-like proteins which have a triple-helical domain of Gly-X-Y amino acid repeats. Detection and quantification of native collagens have been well-established. However, collagen-like proteins differ in their composition and do not have the unique abundance of hydroxyproline and hydroxylysine found in vertebrate collagens. Thus, this poses a problem in the detection and quantification of collagen-like proteins. This paper evaluates reports on the detection and quantification of collagens and collagen-like proteins. A systematic search of the PubMed database was conducted in May 2021, to which five additional papers were added. The 310 unique search results were then subjected to a screening and elimination process, at the end of which 22 papers were included in the study. The findings were summarized and presented in a table that highlights progress in this field. While novel methods have been developed for the detection and quantitation of collagens in general, mainly using enzyme digestion, hybridization, and fluorescence, there is a need for a rapid, one-step method that selectively and sensitively detects and quantitates collagen and collagen-like protein samples with ease.

Elucidation of proteostasis defects caused by osteogenesis imperfecta mutations in the collagen-α2(I) C-propeptide domain

Intracellular collagen assembly begins with the oxidative folding of ∼30-kDa C-terminal propeptide (C-Pro) domains. Folded C-Pro domains then template the formation of triple helices between appropriate partner strands. Numerous C-Pro missense variants that disrupt or delay triple-helix formation are known to cause disease, but our understanding of the specific proteostasis defects introduced by these variants remains immature. Moreover, it is unclear whether or not recognition and quality control of misfolded C-Pro domains is mediated by recognizing stalled assembly of triple-helical domains or by direct engagement of the C-Pro itself. Here, we integrate biochemical and cellular approaches to illuminate the proteostasis defects associated with osteogenesis imperfecta-causing mutations within the collagen-α2(I) C-Pro domain. We first show that “C-Pro-only” constructs recapitulate key aspects of the behavior of full-length Colα2(I) constructs. Of the variants studied, perhaps the most severe assembly defects are associated with C1163R C-Proα2(I), which is incapable of forming stable trimers and is retained within cells. We find that the presence or absence of an unassembled triple-helical domain is not the key feature driving cellular retention versus secretion. Rather, the proteostasis network directly engages the misfolded C-Pro domain itself to prevent secretion and initiate clearance. Using MS-based proteomics, we elucidate how the endoplasmic reticulum (ER) proteostasis network differentially engages misfolded C1163R C-Proα2(I) and targets it for ER-associated degradation. These results provide insights into collagen folding and quality control with the potential to inform the design of proteostasis network-targeted strategies for managing collagenopathies.

Collagen structure: new tricks from a very old dog

The main features of the triple helical structure of collagen were deduced in the mid-1950s from fibre X-ray diffraction of tendons. Yet, the resulting models only could offer an average description of the molecular conformation. A critical advance came about 20 years later with the chemical synthesis of sufficiently long and homogeneous peptides with collagen-like sequences. The availability of these collagen model peptides resulted in a large number of biochemical, crystallographic and NMR studies that have revolutionized our understanding of collagen structure. High-resolution crystal structures from collagen model peptides have provided a wealth of data on collagen conformational variability, interaction with water, collagen stability or the effects of interruptions. Furthermore, a large increase in the number of structures of collagen model peptides in complex with domains from receptors or collagen-binding proteins has shed light on the mechanisms of collagen recognition. In recent years, collagen biochemistry has escaped the boundaries of natural collagen sequences. Detailed knowledge of collagen structure has opened the field for protein engineers who have used chemical biology approaches to produce hyperstable collagens with unnatural residues, rationally designed collagen heterotrimers, self-assembling collagen peptides, etc. This review summarizes our current understanding of the structure of the collagen triple helical domain (COL×3) and gives an overview of some of the new developments in collagen molecular engineering aiming to produce novel collagen-based materials with superior properties.

Collagen XXII binds to collagen-binding integrins via the novel motifs GLQGER and GFKGER

Cell attachment to collagens is mediated by integrins. In the present study, we define two new integrin-binding motifs, GLQGER and GFKGER, within the collagen XXII triple helical domain. Mutation of the two motifs in collagen XXII abolishes the binding to HaCaT cells completely.

Characterization of an Insoluble Collagen Sponge and the Potential for Tissue Engineering Scaffold

Collagen has been widely used in biomedical field, such as scaffolds for tissue engineering. However, the rapidly biodegradation and weak mechanical strength of collagen limited its application. In this study, an insoluble collagen extracted from cattle hide was designed as scaffold to act as a three-dimensional substrate for tissue engineering. The received insoluble collagen sponge was analyzed by scanning electron microscopy, infrared spectroscopy, mechanical testing and thermogravimetric-differential thermal analysis. In addition, the degradation was performed in vitro using collagenase. The results showed that the insoluble collagen had the same triple helical domain as acid-soluble collagen, while the compression strength was greatly improved and the degradation rate was reduced. The insoluble collagen sponge with good stability should be promising in tissue engineering scaffold applications.

Bovine prolactin-related protein-I is anchored to the extracellular matrix through interactions with type IV collagen

The bovine placenta produces an array of proteins structurally similar to pituitary prolactin (PRL). At least ten genes of the bovine placental PRL family, including bovine placental lactogen (bPL) and ten bovine PRL-related protein-I to -X (bPRP-I to -X), encode hormones/cytokines predicted to be involved in the establishment and maintenance of pregnancy. Targets and biological roles for most members of the bovine PRL family have yet to be specified. This study focused on three members of bovine PRL family, bPL, bPRP-I, and bPRP-VI. An alkaline phosphatase (AP) tagging strategy was used to monitor interactions of the ligands with their targets. AP-bPRP-I and AP-bPRP-VI specifically bound to tissue sections of the bovine placentome. AP-bPRP-I and AP-bPRP-VI binding within the placentome mimicked the distribution of the extracellular matrix (ECM). Consequently, AP fusion protein binding to individual ECM components (heparin, laminin, fibronectin, type I collagen, and type IV collagen) was evaluated. AP-bPRP-I specifically bound to type IV collagen, but not to the other ECM components. AP-bPRP-VI exhibited weak interactions with ECM components, while AP-bPL and AP did not show significant binding to any of the ECM components. Binding of AP-bPRP-I to type IV collagen was concentration-dependent, influenced by salt concentrations, and specific to the N-terminal cross-linking domain (7S) of type IV collagen but not its triple-helical domain. The interaction of bPRP-I with type IV collagen suggests that bPRP-I accumulates in the ECM where it likely acts on cells traversing the bovine placentome.