Elsevier

Matrix Biology

Volume 107, March 2022, Pages 77-96
Matrix Biology

O-fucosylation of thrombospondin type 1 repeats is essential for ECM remodeling and signaling during bone development

https://doi.org/10.1016/j.matbio.2022.02.002Get rights and content

Highlights

Deletion of Pofut2 (Protein O-fucosyltransferase 2) in mouse developing limb mesenchyme blocks O-fucosylation of Thrombospondin type-1 repeats (TSR) and significantly shortens limb bones.

Loss of TSR O-fucosylation alters collagen and fibrillins distribution during primary endochondral ossification.

Altered ECM remodeling enhances TGF-β and reduces BMP signaling in developing chondrocytes leading to a reduced hypertrophic zone.

Changes in the ECM composition and signaling likely result from simultaneous reduction in the function and/or level of multiple POFUT2 substrates including members of the ADAMTS family.

Abstract

Many extracellular matrix (ECM) associated proteins that influence ECM properties have Thrombospondin type 1 repeats (TSRs) which are modified with O-linked fucose. The O-fucose is added in the endoplasmic reticulum to folded TSRs by the enzyme Protein O-fucosyltransferase-2 (POFUT2) and is proposed to promote efficient trafficking of substrates. The importance of this modification for function of TSR-proteins is underscored by the early embryonic lethality of mouse embryos lacking Pofut2. To overcome early lethality and investigate the impact of the Pofut2 knockout on the secretion of POFUT2 substrates and on extracellular matrix properties in vivo, we deleted Pofut2 in the developing limb mesenchyme using Prrx1-Cre recombinase. Loss of Pofut2 in the limb mesenchyme caused significant shortening of the limbs, long bones and tendons and stiff joint resembling the musculoskeletal dysplasias in human and in mice with mutations in ADAMTS or ADAMTSL proteins. Limb shortening was evident at embryonic day 14.5 where loss of O-fucosylation led to an accumulation of fibrillin 2 (FBN2), decreased BMP and IHH signaling, and increased TGF-β signaling. Consistent with these changes we saw a decrease in the size of the hypertrophic zone with lower levels of Collagen-X. Unexpectedly, we observed minimal effects of the Pofut2 knockout on secretion of two POFUT2 substrates, CCN2 or ADAMTS17, in the developing bone. In contrast, CCN2 and two other POFUT2 substrates important for bone development, ADAMTS6 and 10, showed a decrease in secretion from POFUT2-null HEK293T cells in vitro. These combined results suggest that the impact of the Pofut2 mutation is cell-type specific. In addition, these observations raise the possibility that the O-fucose modification on TSRs extends beyond promoting efficient trafficking of POFUT2 substrates and has the potential to influence their function in the extracellular environment.

Keywords

Thrombospondin type I repeats
Fucosylation
Fibrillins
TGF-β
Collagen

Abbreviations

ADAMTS
A Disintegrin And Metalloproteinase with ThromboSpondin motifs
ADAMTSL
ADAMTS-Like
ADGRB
Adhesion G protein-coupled receptor B
BAI
Brain-specific angiogenesis inhibitor
BMP
Bone morphogenetic protein
B3GLCT
β3-glucosyltransferase
CCN
Cellular communication network factor-2
CTGF
Connective tissue growth factor
ECM
Extracellular matrix
EDTA
Ethylenediaminetetraacetic acid
EGF
Epidermal growth factor-like
FBN2
Fibrillin 2
FBN1
Fibrillin 1
Gapdh
Glyceraldehyde 3-phosphate dehydrogenase
IHH
Indian hedgehog
LTBP
Latent transforming growth factor beta binding protein
MMP
Matrix metalloproteinase
NOV
Nephroblastoma overexpressed gene
POFUT2
Protein O-fucosyltransferase-2
Rpl4
Ribosomal protein L4
RTN4
Reticulon 4
RUNX2
Runt-related transcription factor 2
SMAD
Sma and Mad related protein
TGF-β
Transforming growth factor-beta
TSP
Thrombospondin
TSR
Thrombospondin type 1 repeat
VEGF
Vascular endothelial growth factor