Alanine-scanning mutations of the BMP-binding domain of recombinant secretory bovine spp24 affect cytokine binding

Abstract

Secreted phosphoprotein 24 kDa (spp24) is a bone morphogenetic protein (BMP)/transforming growth factor-β cytokine-binding protein. The spp24 BMP-2-binding/transforming growth factor receptor II homology-1 (TRH1) domain is a highly conserved N-to-C terminally disulfide-bonded 19-amino acid residue loop similar to those in fetuin and the BMP receptor II. TRH1 domains exhibit a characteristic BTB or β-pleated sheet/turn/β-pleated sheet secondary structure. Our objective was to identify amino acid residues in the spp24 TRH1 domain that bind BMP-2, starting with the nine invariant mammalian residues. Alanine scanning (substitution of Ala for a native residue) was conducted for Cys(110), Arg(111), Ser(112), Thr(113), Val(114), Ser(117), Val(121), Val(124), and Cys(128) of recombinant bovine spp24 (residues 24-203). Binding to rhBMP-2 was assessed by surface plasmon resonance, and the equilibrium binding constants were calculated assuming 1:1 binding between spp24 or its mutants and rhBMP-2, so that affinity = K(D) = k(d)/k(a). Replacing Arg(111) (a positively charged basic residue), polar residues Thr(113) and Ser(117), and the nonpolar Cys(128) with Ala had little effect on BMP-2 binding. Replacing Val(114) or Val(121) with Ala increased binding affinity, whereas replacing Cys(110), Ser(112), Val(124), or both Cys(110) and Cys(128) with Ala decreased it. The kinetics of spp24 binding to BMP-2 can be manipulated by replacing invariant TRH1 residues. Decreasing the relative degree of hydrophobicity in the β-pleated sheet secondary structural motif of the TRH1 domain by replacing key Val residues with Ala increased the affinity for BMP-2 whereas altering the composition of the α-helical turn did not. Thus, the β-pleated sheets play a greater role in BMP-2 binding than the α-helical turn.

Spp24 is associated with endocytic signalling, lipid metabolism, and discrimination of tissue integrity for ‘leaky-gut’ in inflammatory bowel disease

“…This region has been shown to exhibit similarity to the Bone Morphogenic Protein/Transforming Growth Factor-β type II receptor (TRH1 domain) 20 . In addition to bone morphogenic activities as shown by Sun et al 21 ; our data suggests that this protein is modulated in IBD patients 18 , and also has a role in permeability and gastrointestinal epithelial cell integrity. In this study we determine the binding partners of SPP24 and quantitate their ability to differentiate IBD cases of CLE scored variable intestinal permeability in particular UC patients.…”

Secreted phosphoprotein‐24 kDa (Spp24) attenuates BMP‐2‐stimulated Smad 1/5 phosphorylation and alkaline phosphatase induction and was purified in a protective complex with alpha2‐Macroglobulins From Serum

“…TRH1 domains bind cytokines in the BMP/TGFβ superfamily with high affinity and specificity [Demetriou et al, 1996]. The cyclic peptide corresponding to the TRH1 domain of Spp24, designated cyclic BMP‐binding peptide (cBBP), binds rhBMP‐2, BMP‐7, and TGF‐β2 with K D values ranging from 5 to 70 nM [Sun et al, 2010; Taghavi et al, 2010]. Transgenic over‐expression of full‐length (FL) Spp24 in vivo reduces bone mineral density, while FL‐Spp24 inhibits BMP‐2‐stimulated heterotopic bone formation [Sintuu et al, 2008] and BMP‐stimulated spinal fusion [Sintuu et al, 2011].…”

SPP2 Mutations Cause Autosomal Dominant Retinitis Pigmentosa

“…In the SPP2 genes family, bovine spp-24 was the first characterized and suggested to be involved in bone metabolism and fetutin-mineral complex 14 . Subsequently, human Spp-24 was defined with similar structure and was detected in kidney, liver and plasma 9 .…”