Structure-function relationship of collagen-iii mutations in vascular Ehlers-Danlos Syndrome
Creators: name: Gahlawat, Sonal
Publisher: name: No Publisher Supplied
Year: 2024

description: The extracellular matrix (ECM) is a dynamic 3D network essential for tissue development, repair, remodeling, and homeostasis. Collagen, a major structural protein in the ECM, plays a crucial role in maintaining structural integrity and mechanical properties of tissues and organs while regulating critical biological functions. Fibrillar collagens possess a distinctive triple helix structure characterized by a (Gly-X-Y)n repeating sequence. Variations within this sequence can lead to triple helix misfolding, causing heritable connective tissue disorders. The most common variations are single-point missense mutations that replace glycine with a bulkier amino acid (Gly→X).This dissertation aims to elucidate the impact of specific Gly→X substitutions in collagen-III on the tissue structure and function associated with vEDS, a severe, life-threatening condition. Mutations in collagen-III may lead to its abnormal synthesis, secretion, and/or function. Acquiring mutated, full-length collagen with specific mutation from vEDS patients to understand structure-function relationships has proven to be difficult. The discovery of bacterial collagen-like proteins (CLPs) as a model system has enabled systematic investigation of sequence-structure-function relationship in normal and pathological collagens. CLPs self-assemble into a triple helix, similar to animal collagen, and provide a blank template to introduce specific ligand-binding sites and study protein-collagen interactions. In this study, we engineered CLPs with a common integrin-binding site found collagen-III - GFPGER - integrin-binding site (IBS) and introduced Gly→Ser/Arg substitutions either within or outside the IBS. Using a combination of biophysical techniques, enzymatic digestion assays, ELISA, and computational modeling, we demonstrated that Gly→Ser/Arg substitutions differentially impact the triple helix structure, making it susceptible to trypsin digestion, and altering its interaction with integrin receptors, with Arg having a significantly greater impact. Molecular dynamics (MD) and steered MD (SMD) simulations were utilized to explore how Gly→Ser/Arg substitutions affect the triple helix structure and mechanical properties at the molecular level. Arg substitution induced local bulging in the triple helix at the substitution site and disrupted hydrogen bonding compared to Ser substitution. SMD simulations indicated that an Arg substitution led to a substantial reduction in the Young’s modulus of the triple helix.To isolate CLPs containing Gly→X substitutions from full-length constructs, we designed peptides to include a TEV protease cleavage site instead of the commonly used trypsin cleavage site. The triple helix renders collagen resistant to trypsin digestion. CLPs with TEV sites enabled recovery of full-length constructs for both control and Gly→X mutated sequences, whereas Gly→X mutated CLPs with trypsin cleavage sites were fragmented by the enzyme due to the impact of the mutation on the triple helix. CLPs were formed into 3D hydrogels using Michael-type thiol-maleimide addition reactions to present IBS in a native (Gly→X substitutions outside the IBS) versus distorted triple helix (Gly→X substitutions within the IBS). Rheological studies showed that bulkier amino acid substitutions, particularly Gly→Arg substitutions, influenced the shear storage modulus of hydrogels. Cellular assays demonstrated that Gly→Arg substitutions altered human smooth muscle cell behavior, including cell adhesion, spreading, proliferation, and ECM secretion. This findings emphasize the critical role of both the identity and location of the substituted amino acid in determining the loss of function either in terms of cell-matrix interactions and/or mechanical properties. impacting both tissue stiffness and cell function, including ECM production, and tissue remodeling capabilitiesUsing MD simulations, we also investigated the relationship between Gly→X substitutions and their surrounding amino acid sequence. We designed two sequences: a short synthetic peptide with (GPP)16 repeats and a peptide derived from the human collagen-III sequence. Both sequences included a GRPGER IBS with and without Gly→X substitutions. Our results showed that Gly→X substitutions had a more significant impact on the triple helix structure of human collagen-III sequences compared to the highly stable (GPP)16 sequences. Additionally, we demonstrated that capping Gly→X substitutions at different locations with varied imino acid content had differential effects on the triple helix structure. Our findings demonstrate that Gly→X substitutions within imino acid-rich sequences have a lower impact on the triple helix compared to those within imino-poor sequences.Overall, the results presented in this thesis show the impact of Gly→X substitutions on cell-matrix interactions and mechanical properties. This study advances our understanding of mutation-specific effects, utilizing bacterial CLPs to investigate disease mechanisms and guide future therapeutic development for vEDS.