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Transforming growth factor-?2 is sequestered in preterm human milk by chondroitin sulfate proteoglycans.

ABSTRACT: Human milk contains biologically important amounts of transforming growth factor-?2 isoform (TGF-?2), which is presumed to protect against inflammatory gut mucosal injury in the neonate. In preclinical models, enterally administered TGF-?2 can protect against experimental necrotizing enterocolitis, an inflammatory bowel necrosis of premature infants. In this study, we investigated whether TGF-? bioactivity in human preterm milk could be enhanced for therapeutic purposes by adding recombinant TGF-?2 (rTGF-?2) to milk prior to feeding. Milk-borne TGF-? bioactivity was measured by established luciferase reporter assays. Molecular interactions of TGF-?2 were investigated by nondenaturing gel electrophoresis and immunoblots, computational molecular modeling, and affinity capillary electrophoresis. Addition of rTGF-?2 (20-40 nM) to human preterm milk samples failed to increase TGF-? bioactivity in milk. Milk-borne TGF-?2 was bound to chondroitin sulfate (CS) containing proteoglycan(s) such as biglycan, which are expressed in high concentrations in milk. Chondroitinase treatment of milk increased the bioactivity of both endogenous and rTGF-?2, and consequently, enhanced the ability of preterm milk to suppress LPS-induced NF-?B activation in macrophages. These findings provide a mechanism for the normally low bioavailability of milk-borne TGF-?2 and identify chondroitinase digestion of milk as a potential therapeutic strategy to enhance the anti-inflammatory effects of preterm milk.

SUBMITTER: Namachivayam K

PROVIDER: S-EPMC4525106 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Transforming growth factor-β2 is sequestered in preterm human milk by chondroitin sulfate proteoglycans.

Namachivayam Kopperuncholan K Coffing Hayley P HP Sankaranarayanan Nehru Viji NV Jin Yingzi Y MohanKumar Krishnan K Frost Brandy L BL Blanco Cynthia L CL Patel Aloka L AL Meier Paula P PP Garzon Steven A SA Desai Umesh R UR Maheshwari Akhil A

American journal of physiology. Gastrointestinal and liver physiology 20150604 3

Human milk contains biologically important amounts of transforming growth factor-β2 isoform (TGF-β2), which is presumed to protect against inflammatory gut mucosal injury in the neonate. In preclinical models, enterally administered TGF-β2 can protect against experimental necrotizing enterocolitis, an inflammatory bowel necrosis of premature infants. In this study, we investigated whether TGF-β bioactivity in human preterm milk could be enhanced for therapeutic purposes by adding recombinant TGF ...[more]

PMID: 26045614

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Project description: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.

Dataset Information

Transforming growth factor-?2 is sequestered in preterm human milk by chondroitin sulfate proteoglycans.

Publications

Transforming growth factor-β2 is sequestered in preterm human milk by chondroitin sulfate proteoglycans.

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