Abstract
Transforming growth factor-beta (TGF-β), a member of the TGF-β cytokine superfamily, is known to bind to sulfated glycosaminoglycans (GAGs), but the nature of this interaction remains unclear. In a recent study, we found that preterm human milk TGF-β2 is sequestered by chondroitin sulfate (CS) in its proteoglycan form. To understand the molecular basis of the TGF-β2–CS interaction, we utilized the computational combinatorial virtual library screening (CVLS) approach in tandem with molecular dynamics (MD) simulations. All possible CS oligosaccharides were generated in a combinatorial manner to give 24 di- (CS02), 192 tetra- (CS04), and 1536 hexa- (CS06) saccharides. This library of 1752 CS oligosaccharides was first screened against TGF-β2 using the dual filter CVLS algorithm in which the GOLDScore and root-mean-square-difference (RMSD) between the best bound poses were used as surrogate markers for in silico affinity and in silico specificity. CVLS predicted that both the chain length and level of sulfation are critical for the high affinity and high specificity recognition of TGF-β2. Interestingly, CVLS led to identification of two distinct sites of GAG binding on TGF-β2. CVLS also deduced the preferred composition of the high specificity hexasaccharides, which were further assessed in all-atom explicit solvent MD simulations. The MD results confirmed that both sites of binding form stable GAG–protein complexes. More specifically, the highly selective CS chains were found to engage the TGF-β2 monomer with high affinity. Overall, this work present key principles of recognition with regard to the TGF-β2–CS system. In the process, it led to the generation of the in silico library of all possible CS oligosaccharides, which can be used for advanced studies on other protein–CS systems. Finally, the study led to the identification of unique CS sequences that are predicted to selectively recognize TGF-β2 and may out-compete common natural CS biopolymers.
Highlights
Transforming growth factor-beta (TGF-β), a member of the TGF-β superfamily, is known to play important roles in multiple diseases including various cancers, neurodegenerative disorders, and tissue fibrosis
combinatorial virtual library screening (CVLS) led to identification of two distinct sites of GAG binding on TGF-β2
CVLS deduced the preferred composition of the high specificity hexasaccharides, which were further assessed in all-atom explicit solvent molecular dynamics (MD) simulations
Summary
Transforming growth factor-beta (TGF-β), a member of the TGF-β superfamily, is known to play important roles in multiple diseases including various cancers (e.g., colorectal [1], glioblastoma [2], and squamous cell carcinoma [3]), neurodegenerative disorders (e.g., amyotrophic lateral sclerosis [4]), and tissue fibrosis (e.g., skeletal muscle fibrosis [5]). TGF-β1 and TGF-β2 have been the most studied and found to interact with heparin/heparan sulfate (Hp/HS), chondroitin sulfate (CS), and sulfated hyaluronan (sHA) [18,19,20,21,22] Based on these studies, higher levels of sulfation aided by longer chain lengths are two features that engineer higher affinities for TGF-β [16,17], the TGF-β1–HS system has been reported to not follow the oligosaccharide size dependence [19,20]. Despite the importance of TGF-β in human biology and the high possibility of the growth factor’s modulation by GAGs present in the extracellular matrix, atomistic details on the nature of the TGF-β interaction with GAGs are sparse. Our exhaustive computational results support the biophysical studies-based biphasic interaction model reported earlier [21] and bring forth possible avenues for antagonizing or out-competing the biopolymer CS–TGF-β2 system
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