A recombinant technique for mapping functional sites of heterotrimeric collagen helices: Collagen IV CB3 fragment as a prototype for integrin binding.

Autor: Boudko SP; Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA; Center for Matrix Biology, Vanderbilt University Medical Center, Nashville, Tennessee, USA; Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, USA. Electronic address: sergey.budko@vumc.org., Konopka EH; Morphic Therapeutic, Inc, Waltham, Massachusetts, USA., Kim W; Morphic Therapeutic, Inc, Waltham, Massachusetts, USA., Taga Y; Nippi Research Institute of Biomatrix, Toride, Ibaraki, Japan., Mizuno K; Nippi Research Institute of Biomatrix, Toride, Ibaraki, Japan., Springer TA; Department of Biological Chemistry and Molecular Pharmacology, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA., Hudson BG; Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee, USA; Center for Matrix Biology, Vanderbilt University Medical Center, Nashville, Tennessee, USA; Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, USA; Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA; Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, Tennessee, USA; Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee, USA; Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee, USA., Moy TI; Morphic Therapeutic, Inc, Waltham, Massachusetts, USA., Lin FY; Morphic Therapeutic, Inc, Waltham, Massachusetts, USA. Electronic address: albert.lin@morphictx.com.
Jazyk: angličtina
Zdroj: The Journal of biological chemistry [J Biol Chem] 2023 Jul; Vol. 299 (7), pp. 104901. Date of Electronic Publication: 2023 Jun 10.
DOI: 10.1016/j.jbc.2023.104901
Abstrakt: Collagen superfamily of proteins is a major component of the extracellular matrix. Defects in collagens underlie the cause of nearly 40 human genetic diseases in millions of people worldwide. Pathogenesis typically involves genetic alterations of the triple helix, a hallmark structural feature that bestows exceptional mechanical resistance to tensile forces and a capacity to bind a plethora of macromolecules. Yet, there is a paramount knowledge gap in understanding the functionality of distinct sites along the triple helix. Here, we present a recombinant technique to produce triple helical fragments for functional studies. The experimental strategy utilizes the unique capacity of the NC2 heterotrimerization domain of collagen IX to drive three α-chain selection and registering the triple helix stagger. For proof of principle, we produced and characterized long triple helical fragments of collagen IV that were expressed in a mammalian system. The heterotrimeric fragments encompassed the CB3 trimeric peptide of collagen IV, which harbors the binding motifs for α 1 β 1 and α 2 β 1 integrins. Fragments were characterized and shown to have a stable triple helix, post-translational modifications, and high affinity and specific binding of integrins. The NC2 technique is a universal tool for the high-yield production of heterotrimeric fragments of collagens. Fragments are suitable for mapping functional sites, determining coding sequences of binding sites, elucidating pathogenicity and pathogenic mechanisms of genetic mutations, and production of fragments for protein replacement therapy.
Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.
(Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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