TY - JOUR
T1 - Heparin-fibronectin interactions in the development of extracellular matrix insolubility
AU - Raitman, Irene
AU - Huang, Mia L.
AU - Williams, Selwyn A.
AU - Friedman, Benjamin
AU - Godula, Kamil
AU - Schwarzbauer, Jean E.
N1 - Funding Information:
This research was supported by the National Institutes of Health (NIH) [ R01 CA160611 to J.E.S.]. IR was supported by NIH pre-doctoral training grant T32 GM007388 to Princeton University Department of Molecular Biology. SAW was supported by NIH NRSA fellowship F32 GM077891. MLH and KG were supported in part by the NIH Pathway to Independence Award [NIBIB: 4 R00 EB013446], the NIH Director's New Innovator Award [1DP2HD087954], and the Program of Excellence in Glycosciences [PEG, NHLBI: 4P01HL107150].
Publisher Copyright:
© 2017 Elsevier B.V.
PY - 2018/4
Y1 - 2018/4
N2 - During extracellular matrix (ECM) assembly, fibronectin (FN) fibrils are irreversibly converted into a detergent-insoluble form which, through FN's multi-domain structure, can interact with collagens, matricellular proteins, and growth factors to build a definitive matrix. FN also has heparin/heparan sulfate (HS) binding sites. Using HS-deficient CHO cells, we show that the addition of soluble heparin significantly increased the amount of FN matrix that these cells assemble. Sulfated HS glycosaminoglycan (GAG) mimetics similarly increased FN assembly and demonstrated a dependence on GAG sulfation. The length of the heparin chains also plays a role in assembly. Chains of sufficient length to bind to two FN molecules gave maximal stimulation of assembly whereas shorter heparin had less of an effect. Using a decellularized fibroblast matrix for proteolysis, detergent fractionation, and mass spectrometry, we found that the predominant domain within insoluble fibril fragments is FN's major heparin-binding domain HepII (modules III12–14). Multiple HepII domains bind simultaneously to a single heparin chain in size exclusion chromatography analyses. We propose a model in which heparin/HS binding to the HepII domain connects multiple FNs together to facilitate the formation of protein interactions for insoluble fibril assembly.
AB - During extracellular matrix (ECM) assembly, fibronectin (FN) fibrils are irreversibly converted into a detergent-insoluble form which, through FN's multi-domain structure, can interact with collagens, matricellular proteins, and growth factors to build a definitive matrix. FN also has heparin/heparan sulfate (HS) binding sites. Using HS-deficient CHO cells, we show that the addition of soluble heparin significantly increased the amount of FN matrix that these cells assemble. Sulfated HS glycosaminoglycan (GAG) mimetics similarly increased FN assembly and demonstrated a dependence on GAG sulfation. The length of the heparin chains also plays a role in assembly. Chains of sufficient length to bind to two FN molecules gave maximal stimulation of assembly whereas shorter heparin had less of an effect. Using a decellularized fibroblast matrix for proteolysis, detergent fractionation, and mass spectrometry, we found that the predominant domain within insoluble fibril fragments is FN's major heparin-binding domain HepII (modules III12–14). Multiple HepII domains bind simultaneously to a single heparin chain in size exclusion chromatography analyses. We propose a model in which heparin/HS binding to the HepII domain connects multiple FNs together to facilitate the formation of protein interactions for insoluble fibril assembly.
KW - Extracellular matrix
KW - Fibronectin
KW - Glycosaminoglycans
KW - Heparin
KW - Matrix assembly
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U2 - 10.1016/j.matbio.2017.11.012
DO - 10.1016/j.matbio.2017.11.012
M3 - Article
C2 - 29223498
AN - SCOPUS:85039046845
SN - 0945-053X
VL - 67
SP - 107
EP - 122
JO - Collagen and Related Research
JF - Collagen and Related Research
ER -