TY - JOUR
T1 - Glycosylation oftwisted gastrulation is required for BMP binding and activity during craniofacial development
AU - Billington, Charles J.
AU - Fiebig, Juliane E.
AU - Forsman, Cynthia L.
AU - Pham, Lan
AU - Burbach, Nathan
AU - Sun, Mu
AU - Jaskoll, Tina
AU - Mansky, Kim
AU - Gopalakrishnan, Rajaram
AU - O'Connor, Michael B.
AU - Mueller, Thomas D.
AU - Petryk, Anna
PY - 2011
Y1 - 2011
N2 - Twisted gastrulation (TWSG1) is a conserved, secreted glycoprotein that modulates signaling of bone morphogenetic proteins (BMPs) in the extracellular space. Deletion of exon 4 of mouse Twsg1 (mTwsg1) is associated with significant craniofacial defects. However, little is understood about the biochemical properties of the corresponding region of the protein. We have uncovered a significant role for exon 4 sequences as encoding the only two glycosylation sites of the mTWSG1 protein. Deletion of the entire exon 4 or mutation of both glycosylation sites within exon 4 abolishes glycosylation of mTWSG1. Importantly, we find that constructs with mutated glycosylation sites have significantly reduced BMP binding activity. We further show that glycosylation and activity of TWSG1 recombinant proteins vary markedly by cellular source. Non-glycosylated mTWSG1 made in E. coli has both reduced affinity for BMPs, as shown by surface plasmon resonance analysis, and reduced BMP inhibitory activity in a mandibular explant culture system compared to gly-cosylated proteins made in insect cells or murine myeloma cells. This study highlights an essential role for glycosylation in Twisted gastrulation action.
AB - Twisted gastrulation (TWSG1) is a conserved, secreted glycoprotein that modulates signaling of bone morphogenetic proteins (BMPs) in the extracellular space. Deletion of exon 4 of mouse Twsg1 (mTwsg1) is associated with significant craniofacial defects. However, little is understood about the biochemical properties of the corresponding region of the protein. We have uncovered a significant role for exon 4 sequences as encoding the only two glycosylation sites of the mTWSG1 protein. Deletion of the entire exon 4 or mutation of both glycosylation sites within exon 4 abolishes glycosylation of mTWSG1. Importantly, we find that constructs with mutated glycosylation sites have significantly reduced BMP binding activity. We further show that glycosylation and activity of TWSG1 recombinant proteins vary markedly by cellular source. Non-glycosylated mTWSG1 made in E. coli has both reduced affinity for BMPs, as shown by surface plasmon resonance analysis, and reduced BMP inhibitory activity in a mandibular explant culture system compared to gly-cosylated proteins made in insect cells or murine myeloma cells. This study highlights an essential role for glycosylation in Twisted gastrulation action.
KW - BMP
KW - Glycosylation
KW - Mandibular explants
KW - Msx2
KW - Surface plasmon resonance analysis
KW - Twisted gastrulation
UR - http://www.scopus.com/inward/record.url?scp=84866180783&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84866180783&partnerID=8YFLogxK
U2 - 10.3389/fphys.2011.00059
DO - 10.3389/fphys.2011.00059
M3 - Article
C2 - 21941513
AN - SCOPUS:84866180783
SN - 1664-042X
VL - 2 SEP
JO - Frontiers in Physiology
JF - Frontiers in Physiology
M1 - Article 59
ER -