Consequences of Glycine Mutations in the Fibronectin-binding Sequence of Collagen

Chhum, Panharith; Yu, Hongtao; An, Bo; Doyon, Brian; Lin, Yu‐Shan; Brodsky, Barbara

doi:10.1074/jbc.m116.753566

Cited by 21 publications

(22 citation statements)

References 56 publications

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“…Recent studies indicate the importance of the glycine substitutions in changing stability of the triple helix and interactions of collagen with other proteins of extracellular matrix like fibronectin etc. (Ito et al, ; Chhum et al, ). Mutations of COL2A1 may result in conditions other than skeletal dysplasias (Kannu, Bateman, Belluoccio, Fosang, & Savarirayan, ).…”

Section: Discussionmentioning

confidence: 99%

Exome sequencing reveals a novel COL2A1 mutation implicated in multiple epiphyseal dysplasia

Dasa

Eastwood

Podgórski

et al. 2019

American J of Med Genetics Pt A

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Mutations in the COMP, COL9A1, COL9A2, COL9A3, MATN3, and SLC26A2 genes cause approximately 70% of multiple epiphyseal dysplasia (MED) cases. The genetic changes involved in the etiology of the remaining cases are still unknown, suggesting that other genes contribute to MED development. Our goal was to identify a mutation causing an autosomal dominant form of MED in a large multigenerational family. Initially, we excluded all genes known to be associated with autosomal dominant MED by using microsatellite and SNP markers. Follow-up with wholeexome sequencing analysis revealed a mutation c.2032G>A (p.Gly678Arg) in the COL2A1 gene (NCBI Reference Sequence: NM_001844.4), which co-segregated with the disease phenotype in this family, manifested by severe hip dysplasia and osteoarthritis. One of the affected family members had a double-layered patella, which is frequently seen in patients with autosomal recessive MED caused by DTDST mutations and sporadically in the dominant form of MED caused by COL9A2 defect. K E Y W O R D Sdouble-layered patella, multiple epiphyseal dysplasia, novel mutation in COL2A1

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Section: Discussionmentioning

confidence: 99%

Exome sequencing reveals a novel COL2A1 mutation implicated in multiple epiphyseal dysplasia

Dasa

Eastwood

Podgórski

et al. 2019

American J of Med Genetics Pt A

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“…Previous studies have shown that substitution of these integral Gly residues causes structural deformities to the triple helix, and the extent of triple-helix destabilization depends on the amino acid substituted (30,31) and the surrounding sequence context (32)(33)(34)(35)(36). Recently, elegant studies have investigated the impact of Gly 3 Xaa mutations near integrinbinding sites in collagen I (31, 37) using recombinant bacterial collagen systems, which can incorporate long spans of collagenlike sequence and can probe selective mutation sites.…”

mentioning

confidence: 99%

Molecular underpinnings of integrin binding to collagen-mimetic peptides containing vascular Ehlers–Danlos syndrome–associated substitutions

Hoop

Kemraj

Wang

et al. 2019

Journal of Biological Chemistry

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Collagens carry out critical extracellular matrix (ECM) functions by interacting with numerous cell receptors and ECM components. Single glycine substitutions in collagen III, which predominates in vascular walls, result in vascular Ehlers-Danlos syndrome (vEDS), leading to arterial, uterine, and intestinal rupture and an average life expectancy of <50 years. Collagen interactions with integrin ␣ 2 ␤ 1 are vital for platelet adhesion and activation; however, how these interactions are impacted by vEDS-associated mutations and by specific amino acid substitutions is unclear. Here, we designed collagen-mimetic peptides (CMPs) with previously reported Gly 3 Xaa (Xaa ‫؍‬ Ala, Arg, or Val) vEDS substitutions within a high-affinity integrin ␣ 2 ␤ 1binding motif, GROGER. We used these peptides to investigate, at atomic-level resolution, how these amino acid substitutions affect the collagen III-integrin ␣ 2 ␤ 1 interaction. Using a multitiered approach combining biological adhesion assays, CD, NMR, and molecular dynamics (MD) simulations, we found that these substitutions differentially impede human mesenchymal stem cell spreading and integrin ␣ 2-inserted (␣ 2 I) domain binding to the CMPs and were associated with triple-helix destabilization. Although an Ala substitution locally destabilized hydrogen bonding and enhanced mobility, it did not significantly reduce the CMP-integrin interactions. MD simulations suggested that bulkier Gly 3 Xaa substitutions differentially disrupt the CMP-␣ 2 I interaction. The Gly 3 Arg substitution destabilized CMP-␣ 2 I side-chain interactions, and the Gly 3 Val change broke the essential Mg 2؉ coordination. The relationship between the loss of functional binding and the type of vEDS substitution provides a foundation for developing potential therapies for managing collagen disorders.

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“…The synthetic peptides and the phosphorylation products were analyzed with a MALDI-TOF Microflex LT instrument (Bruker, Billerica, MA) in positive ion mode. Samples were mixed with MALDI matrix (a saturated sinapinic acid solution in 50% (v/v) acetonitrile and 0.3% (v/v) trifluoroacetic acid) in a 1:4 ratio, and 1 mL of the peptide-containing matrix was pipetted onto a MALDI target plate and allowed to dry, as previously described (20). It was noted that each peptide produced multiple peaks, one at the expected mass of the peptide as well as a second peak at a higher molecular weight that corresponded to the mass of the peptide plus an associated cation (either sodium or potassium).…”

Section: Mass Spectrometrymentioning

confidence: 99%

“…MD simulations were carried out to investigate whether the bulky and charged pSer within the Gly-Xaa-Yaa repeating sequence of peptide pSer546 could be incorporated into a standard triple helix. The sequence of the phosphorylated peptide was constructed into a triple helix (20). MD simulations were carried out for 100 ns at 300 K using the Amber99SB force field and TIP3P water model, following a previously described simulation protocol (3,20).…”

Section: Simulations Of the Collagen Triple Helix With Psermentioning

confidence: 99%

“…The sequence of the phosphorylated peptide was constructed into a triple helix (20). MD simulations were carried out for 100 ns at 300 K using the Amber99SB force field and TIP3P water model, following a previously described simulation protocol (3,20). At neutral pH, the phosphate group of pSer is fully deprotonated, with a À2e charge, and the Asp side chain in the triplet following the GSP has a À1e charge, raising the possibility of repulsive electrostatic interactions between them.…”

Section: Simulations Of the Collagen Triple Helix With Psermentioning

confidence: 99%

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Enzymatic Phosphorylation of Ser in a Type I Collagen Peptide

Qiu

Poppleton

Mekkat

et al. 2018

Biophysical Journal

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Phosphoproteomics studies have reported phosphorylation at multiple sites within collagen, raising the possibility that these post-translational modifications regulate the physical or biological properties of collagen. In this study, molecular dynamics simulations and experimental studies were carried out on model peptides to establish foundational principles of phosphorylation of Ser residues in collagen. A (Gly-Xaa-Yaa) 11 peptide was designed to include a Ser-containing sequence from type I collagen that was reported to be phosphorylated. The physiological kinase involved in collagen phosphorylation is not known. In vitro studies showed that a model kinase ERK1 (extracellular signal-regulated protein kinase 1) would phosphorylate Ser within the consensus sequence if the collagen-like peptide is in the denatured state but not in the triple-helical state. The peptide was not a substrate for FAM20C, a kinase present in the secretory pathway, which has been shown to phosphorylate many extracellular matrix proteins. The unfolded single chain (Gly-Xaa-Yaa) 11 peptide containing phosphoSer was able to refold to form a stable triple helix but at a reduced folding rate and with a small decrease in thermal stability relative to the nonphosphorylated peptide at neutral pH. These biophysical studies on model peptides provide a basis for investigations into the physiological consequences of collagen phosphorylation and the application of phosphorylation to regulate the properties of collagen biomaterials.

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Consequences of Glycine Mutations in the Fibronectin-binding Sequence of Collagen

Cited by 21 publications

References 56 publications

Exome sequencing reveals a novel COL2A1 mutation implicated in multiple epiphyseal dysplasia

Exome sequencing reveals a novel COL2A1 mutation implicated in multiple epiphyseal dysplasia

Molecular underpinnings of integrin binding to collagen-mimetic peptides containing vascular Ehlers–Danlos syndrome–associated substitutions

Enzymatic Phosphorylation of Ser in a Type I Collagen Peptide

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