TY - JOUR
T1 - Betaglycan has two independent domains required for high affinity TGF-β binding
T2 - Proteolytic cleavage separates the domains and inactivates the neutralizing activity of the soluble receptor
AU - Mendoza, Valentín
AU - Vilchis-Landeros, M. Magdalena
AU - Mendoza-Hernández, Guillermo
AU - Huang, Tao
AU - Villarreal, Maria M.
AU - Hinck, Andrew P.
AU - López-Casillas, Fernando
AU - Montiel, Jose Luis
PY - 2009/12/15
Y1 - 2009/12/15
N2 - Betaglycan is a coreceptor for members of the transforming growth factor β (TGF-β) superfamily. Mutagenesis has identified two ligand binding regions, one at the membrane-distal and the other at the membrane-proximal half of the betaglycan ectodomain. Here we show that partial plasmin digestion of soluble betaglycan produces two proteolysis-resistant fragments of 45 and 55 kDa, consistent with the predicted secondary structure, which indicates an intervening nonstructured linker region separating the highly structured N- and C-terminal domains. Amino terminal sequencing indicates that the 45 and 55 kDa fragments correspond, respectively, to the membrane-distal and -proximal regions. Plasmin treatment of membrane betaglycan results in the production of equivalent proteolysis-resistant fragments. The 45 and 55 kDa fragments, as well as their recombinant soluble counterparts, Sol Δ10 and Sol Δ11, bind TGF-β, but nonetheless, compared to intact soluble betaglycan, have a severely diminished ability to block TGF-β activity. Surface plasmon resonance (SPR) analysis indicates that soluble betaglycan has Kd's in the low nanomolar range for the three TGF-β isoforms, while those for Sol Δ10 and Sol Δ11 are 1-2 orders of magnitude higher. SPR analysis further shows that the Kd's of Sol Δ11 are not changed in the presence of Sol Δ10, indicating that the high affinity of soluble betaglycan is a consequence of tethering the domains together. Overall, these results suggest that betaglycan ectodomain exhibits a bilobular structure in which each lobule folds independently and binds TGF-β through distinct nonoverlapping interfaces and that linker modification may be an approach to improve soluble betaglycan's TGF-β neutralizing activity.
AB - Betaglycan is a coreceptor for members of the transforming growth factor β (TGF-β) superfamily. Mutagenesis has identified two ligand binding regions, one at the membrane-distal and the other at the membrane-proximal half of the betaglycan ectodomain. Here we show that partial plasmin digestion of soluble betaglycan produces two proteolysis-resistant fragments of 45 and 55 kDa, consistent with the predicted secondary structure, which indicates an intervening nonstructured linker region separating the highly structured N- and C-terminal domains. Amino terminal sequencing indicates that the 45 and 55 kDa fragments correspond, respectively, to the membrane-distal and -proximal regions. Plasmin treatment of membrane betaglycan results in the production of equivalent proteolysis-resistant fragments. The 45 and 55 kDa fragments, as well as their recombinant soluble counterparts, Sol Δ10 and Sol Δ11, bind TGF-β, but nonetheless, compared to intact soluble betaglycan, have a severely diminished ability to block TGF-β activity. Surface plasmon resonance (SPR) analysis indicates that soluble betaglycan has Kd's in the low nanomolar range for the three TGF-β isoforms, while those for Sol Δ10 and Sol Δ11 are 1-2 orders of magnitude higher. SPR analysis further shows that the Kd's of Sol Δ11 are not changed in the presence of Sol Δ10, indicating that the high affinity of soluble betaglycan is a consequence of tethering the domains together. Overall, these results suggest that betaglycan ectodomain exhibits a bilobular structure in which each lobule folds independently and binds TGF-β through distinct nonoverlapping interfaces and that linker modification may be an approach to improve soluble betaglycan's TGF-β neutralizing activity.
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U2 - 10.1021/bi901528w
DO - 10.1021/bi901528w
M3 - Article
C2 - 19842711
AN - SCOPUS:71549129301
VL - 48
SP - 11755
EP - 11765
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 49
ER -