Abstract
The human fetus and neonate swallow biologically significant quantities of IL-8/CXC ligand 8 (CXCL8) in amniotic fluid and breast milk, and this remains measurable through simulated neonatal gastric and proximal intestinal digestions. We sought to confirm the structural and functional integrity of IL-8/CXCL8 in digestates and determine the mechanisms underlying this protease resistance. We observed that in comparison with BSA, IL-8/CXCL8 is highly resistant to pepsin and can be detected intact in assays for structural, immunologic, and functional integrity. In a computational molecular docking simulation, IL-8/CXCL8 was observed to fit poorly in the pepsin active site. On the basis of simulated mutation analyses, we hypothesized that this protease resistance is due to disulfide bond-related tertiary folding in IL-8/CXCL8. This was confirmed on chemical reduction of these groups.
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Abbreviations
- CXCL8:
-
CXC ligand 8
- FIRST:
-
Floppy Inclusion and Rigid Substructure Topography
- IEC:
-
intestinal epithelial cell
- PDB:
-
protein data bank
- rhIL-8/CXCL8:
-
recombinant human IL-8/CXCL8
- SGF:
-
simulated gastric fluid
- TCEP:
-
Tris [2-carboxyethyl] phosphine hydrochloride
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Acknowledgements
We thank Drs. D.J. Jacobs, Leslie A. Kuhn, and M.F. Thorpe (Michigan State University, East Lansing, MI) for permission to use the algorithm FIRST, and Dr. Gert Vriend (University of Groningen, The Netherlands) for WHAT IF. We are grateful to Nicolas Guex, Alexandre Diemand, Manuel C. Peitsch, Torsten Schwede, and GlaxoSmithKline R&D for allowing access to the Swiss deep view pdb viewer (http://www.expasy.org/spdbv) and to the Nussinov-Wolfson Structural Bioinformatics Group (Tel-Aviv University, Israel) for allowing access to the FlexProt protein alignment algorithm. We thank Drs. Deepak Agrawal (Moffitt Cancer Center, Tampa, FL) and Jane Carver for thoughtful discussions on the manuscript; Dawn Bruton, Sue Stripling, and Debbie Patrick, RN, for help with sample and data collection; and Ingrid Williams for managerial support.
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This work was supported by grants HD-01180, HD-42326, and HL-61798 from the National Institutes of Health.
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Maheshwari, A., Lu, W., Guida, W. et al. IL-8/CXC Ligand 8 Survives Neonatal Gastric Digestion as a Result of Intrinsic Aspartyl Proteinase Resistance. Pediatr Res 57, 438–444 (2005). https://doi.org/10.1203/01.PDR.0000151317.08180.7E
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DOI: https://doi.org/10.1203/01.PDR.0000151317.08180.7E
