BMP6 binding to heparin and heparan sulfate is mediated by N-terminal and C-terminal clustered basic residues

https://doi.org/10.1016/j.bbagen.2020.129799Get rights and content

Highlights

  • Arginine- and Lysine-rich regions were identified on BMP6 as potential heparin/HS-binding domains (HBDs).

  • BMP6 peptides and recombinant protein bound immobilized heparin and HS.

  • We dissected two heparin binding sites on BMP6: the N-terminal R5 R6 R7 and the C-terminal K126 K127 R129.

  • In in silico study the N-terminus flexibly adapted to heparin and the C-terminus strongly contributed to binding energy.

Abstract

Background

The bone morphogenetic protein 6 (BMP6) is a crucial inducer of hepcidin, the peptide hormone that regulates the iron availability in our body. Hepcidin expression is influenced by hepatic heparan sulfate (HS) and by heparin administration, suggesting BMP6 interaction with heparin/HS. The BMP2/4 subfamily has been deeply characterized to have a N-terminal heparin/HS binding domain (HBD), whose basic residues contact the sulfate groups on heparin and HS. Such detailed characterization is still required for other, structurally different BMPs, including BMP6.

Methods

BMP6 peptides encompassing potential HBDs were analysed on heparin-functionalized plates and microcantilevers, and on membrane HS expressing CHO-K1 cells. Monomeric wild-type BMP6 and mutants were produced, substituting the basic residues with non-charged ones, and their affinity to the heparin-column was measured. The BMP6-heparin interaction was also predicted at atomic level by in silico molecular dynamics.

Results

N-terminal and C-terminal BMP6 peptides showed high heparin affinity in solid-phase assays. The mutation of the two sites (R5L, R6S, R7L and K126N, K127N, R129S) abolished the heparin-binding activity of the recombinant monomeric BMP6. Monomeric BMP6 and peptides specifically bound to membrane HS of CHO-K1 cells through the same domains. Molecular dynamic studies supported the role of the two HBDs, suggesting a cooperative behaviour.

Conclusions

In BMP6, N-terminal (R5, R6, R7) and C-terminal (K126, K127, R129) domains mediate the interaction with heparin and HS.

General significance

This study provides the molecular mechanism supporting the use of heparin to sequester BMP6 and inhibit hepcidin expression, a novel clinical approach for high-hepcidin iron disorders.

Introduction

The bone morphogenetic proteins (BMPs) represent the largest subgroup of the transforming growth factor-β superfamily, that includes over 20 members in human [8]. They are all synthesized as pro-protein precursors undergoing proteolytic cleavage to generate the mature chains that then assemble into dimers, functional in binding the BMP receptors to elicit cellular signalling. After binding the ligand, the receptors complex phosphorylates the downstream effectors SMAD1/5/8, which then associate with SMAD4 and translocate into the nucleus to induce the expression of several genes [25,26,58]. Despite the common mechanism of signalling, BMPs are involved in different biological processes of development and homeostasis, beyond the osteogenic function that was firstly described [56]. BMPs activities can be modulated by other molecules, among which the heparan sulfate (HS) glycans, that were shown to influence the spatial control and protein-protein interactions of some BMPs [51].

All the animal cells express heparan sulfate proteoglycans (HSPGs), glycoproteins that contain one or more HS polysaccharide chains linked to the proteoglycan protein core [7,38]. The HS chain is made of repeated disaccharidic units of N-acetyl-glucosamine (GlcNAc) and glucuronic acid (GlcA), linked by a 1–4 glycosidic bond. During their assembly, the HS chains undergo a series of processing reactions involving GlcNAc N-deacetylation and N-sulfation, epimerization of GlcA to iduronic acid (IdoA), and O-sulfation that generate relatively short segments of modified sugars interspersed by variable tracts of unmodified sugars [15]. Heparin is considered part of this group of glycans, although it is an unusually short and highly sulfated HS chain [37].

BMPs were initially purified on heparin columns, but the interaction with heparin/HS has been well-characterized mainly for BMP2 and BMP4: the heparin/HS-binding domains (HBDs) were identified as N-terminal cores rich in basic residues, that contact the negatively charged sulfate groups displayed by heparin/HS [42,52]. This interaction is biologically relevant, since heparin prevents BMP2 binding to BMP receptors inhibiting the osteogenic activity [33], while membrane-bound HS facilitates the BMP2-induced BMP receptor complex formation in vitro [35] and regulates BMP4 action range in Xenopus embryo [42].

BMP6 belongs to a BMP subfamily different from that of BMP2/4 and its HBDs are yet to be established [51]. BMP6 plays a major role in bone formation [56] but it is also a key player in systemic iron homeostasis, since it is the major inducer of the hepatic expression of the peptide hormone hepcidin that regulates systemic iron availability [1,10]. Interestingly, exogenous heparin administration inhibits BMP6 biological activities both as osteogenic factor [9] and as hepcidin inducer [3,[45], [46], [47], [48]], while the alteration of endogenous hepatic HS specifically downregulates the BMP/SMAD signalling and hepcidin expression [4,49]. Altogether these observations showed that a glycan-protein interaction influences BMP6 function. The difficulties to produce the full-length BMP6 as soluble recombinant folded protein [54] likely dampened detailed studies of its interaction with heparin/HS. Consequently only a recent report by Billings et al. investigated this aspect using BMP6 peptides that led to the identification of a novel HBD located near the C-terminus [6].

In our present work, we investigated the BMP6 activity in binding heparin and HS using different approaches. We identified clusters of basic residues as putative interaction sites and the corresponding synthetic peptides were studied for heparin and HS binding in solid-phase assays to identify two major domains. Then, the full-length human monomeric BMP6 was expressed in E. coli and assessed to interact with heparin and HS. The substitution of basic residues with neutral ones in two putative HBDs, located near the C-terminus and the N-terminus, strongly reduced the heparin interaction. In addition, in silico study of BMP6-heparin interaction confirmed the in vitro observation and proposed the atomic details of possible molecular recognition and contact sites among BMP6 and a representative heparin chain. The results confirm and extend the recent findings by Billings and colleagues [6].

Section snippets

BMP6 peptides

Linear peptides, corresponding to BMP6 arginine and lysine rich domains, were synthesized by Thermo Fisher Scientific custom peptide service (Table 1). A N-terminal biotin tag was linked to each peptide, allowing the detection during the binding in vitro assays.

Heparin-coated plate assay

Immunosorbent 96-well plate (immunoGRADE, BRANDplates®) was coated with 10 μg/mL protamine salt from salmon (no. P4020, Sigma) in 50 mM carbonate buffer pH 9.6, overnight at 4 °C, generating a first cationic-polypeptide coating layer for

Identification of heparin/HS-binding domains in the BMP6 sequence

The interaction of proteins with heparin/HS is mainly mediated by clusters of positive charged arginine and lysine residues that attract the negatively charged sulfate groups of heparin/HS [57]. In order to identify conserved basic residues cores, we aligned the mature BMP6 sequence from different animal species. Three conserved domains were identified, representing putative BMP6 heparin/HS-binding domains (HBDs) (Fig. 1).

These domains were localized near the N-terminus (HBD1), in central

Conclusions

The BMP6 interaction with heparin and HS was initially suggested by the observations that exogenous heparin inhibits the BMP6-induced hepcidin expression, while hepatic HS facilitate BMP6 activity [3,[45], [46], [47], [48], [49]]. However, a biochemical characterization of BMP6 interaction with this family of sulfated glycans was missing until recently, when Billings et al. in 2018 using various peptides showed that BMP6 binds HS with high affinity with a domain at C-terminus, differently by

Authorship

A.D.: designed research, performed research, analysed data and wrote the paper;

SE.: performed the in-silico studies and contributed to write the paper;

S.F.: performed the Microcantilever studies and revised the paper;

M.A., M.G., P.R., F.C.: performed research and revised the paper;

P.A.: contributed to design of research and revised the paper.

P.B., A.N: revised the paper.

M.P.: designed research, analysed data and wrote the paper.

Declaration of Competing Interest

The authors declare no competing financial interests.

Acknowledgements

We are grateful to Dr. Emiliano Esposito from G. Ronzoni Institute, Milan for the preparation of thiolated heparin. The work was partially supported by M.P. with ex60% from the University of Brescia, Brescia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The work of S. E. was supported by G. Ronzoni Institute, Milan.

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