George R. Bousfield PhD

Overview

Director of the Protein Chemistry Core Laboratory.

Areas of Research Interest

Research Interests

My laboratory is interested in the structure-function relations of the gonadotropins. These are members of the glycoprotein hormone family, which consists of the three gonadotropins, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and chorionic gonadotropin (CG). The fourth member of this protein family is thyroid stimulating hormone (TSH). Together, the glycoprotein hormones form a unique subset of the cystine knot growth factor superfamily. While most other superfamily members are located at the C-terminal end of a larger precursor from which they are cleaved, and the N-terminal portion then acts as a regulatory factor, the glycoprotein subunits consist of only the cystine knot domains. The subunits are dissimilar proteins, a common alpha subunit that is differentially glycosylated in each hormone, as well as a hormone-specific beta subunit. Another unusual facet of glycoprotein hormone structure-function is these hormones are decorated with N-linked and sometimes O-linked glycans, which are necessary for function. The glycans can determine metabolic clearance rates of the hormones and at least some of them are necessary for biological activity, although the mechanism is unknown. Glycosylation varies in two ways: microheterogeneity and macroheterogeneity. The former results from a population of 2-105 glycans found at each N- or O-glycosylation site, while the latter results from partial glycosylation of the hormone-specific beta subunit. In humans an interesting pattern of glycosylation is found in FSH. The alpha subunit is always N-glycosylated, while the FSHΒ subunit appears to be glycosylated in an all-or-none manner. This produces a FSH glycoform that possesses four N-glycans that we call tetra-glycosylated hFSH and another form that possesses only the alpha subunit N-glycans that we call di-glycosylated hFSH. In young women, di-glycosylated hFSH is the more abundant glycoform present in the pituitary gland, however, there appears to be a progressive loss of this glycoform with increasing age. We are interested in determining if this can be used as a marker for reproductive aging.

Areas of Teaching Interest

Courses Taught

Publications

Publications

1. Teng, C.T., Teng, C-S., Bousfield, G.R., Liu, W-K., & Ward, D.N. 1982. Differential Response  of Growing and Regressing Chicken Ovaries to Gonadotropic Hormones.  Gen. Comp. Endocrinol., 48, 325-332.
2. Jirgensens, B., Bousfield, G.R., & Moore, W.T., Jr. 1983. Circular Dichroism and Conformational Properties of Modecin, A Toxic Protein.  J. Protein Chem., 2, 105-112.
3. Bousfield, G.R., & Ward, D.N. 1984. Purification of Lutropin and Follitropin in High Yield from Horse Pituitary Glands.  J. Biol. Chem.,  259, 1911-1921.
4. Bousfield, G.R., Liu, W-K., & Ward, D.N. 1985. Hybrids from equine LH: alpha enhances, beta diminishes activity.  Molec. Cell. Endocrinol.,  40, 60-77.
5. Bousfield, G.R., Sugino, H., & Ward, D.N. 1985. Demonstration of a COOH-terminal Extension on Equine Lutropin by Means of a Common Acid-labile Bond in Equine Lutropin and Chorionic Gonadotropin.  J. Biol. Chem., 260, 9531-9533.
6. Liu, W-K., Bousfield, G.R., Moore, W.T., Jr., & Ward, D.N. 1985. Priming Procedure and  Hormone Preparations Influence Rat Granulosa Cell Response.  Endocrinology, 116, 1454-1459.
7. Bousfield, G.R. & Ward, D.N. 1986. Direct Demonstration of Intrinsic Follicle-Stimulating Hormone Receptor-Binding Activity in Acid-treated Equine Luteinizing Hormone.  Biochim. Biophys. Acta., 885, 327-334.
8. Liehr, J.G., Stancel, G.M., Chorich, L.P., Bousfield, G.R., & Ulubelen, A.A. 1986. Hormonal Carcinognesis:  Separation of Estrogenicity from Carcinogenicity.  Chem.-Biol. Interactions, 59, 173-184.
9. Sugino, H., Bousfield, G.R., Moore, W.T., Jr., & Ward, D.N. 1987. Structural Studies on Equine Glycoprotein Hormones:  Amino acid Sequence of Equine Chorionic Gonadotropin Beta-Subunit.  J. Biol. Chem.,  262, 8603-8609.
10. Bousfield, G.R., Liu, W-K., Sugino, H., & Ward, D.N. 1987. Structural Studies on Equine Glycoprotein Hormones:  Amino acid Sequence of Equine Lutropin Beta Subunit.  J. Biol. Chem.,  262, 8610-8620.
11. Bidart, J-M., Troalen, F., Salesse, Bousfield, G.R., Bohouon, C.J., & Bellet, D.H. 1987. Topographic Antigenic Determinants Recognized by Monoclonal Antibodies on Human Choriogonadotropin Beta-Subunit.  J. Biol. Chem., 262, 8551-8556.
12. Ward, D.N., Wen, T., & Bousfield, G.R. 1987. Sulfate and Phosphate Analysis in Glycoproteins and Other Biologic Compounds Using Ion Chromatography:  Application to Glycoprotein Hormones and Sugar Esters.  J. Chromatog.,  398,  255-264.
13. Bidart, J-M., Troalen, F., Bousfield, G.R., Birken, S., & Bellet, D. 1988. Antigenic Determinants on Human Choriogonadotropin Beta-Subunit.  I.  Characterization of Topographic Sites Recognized by Monoclonal Antibodies.  J. Biol. Chem.,  263, 10364-10369.
14. Bousfield, G.R. & Ward, D.N. 1988. Selective Proteolysis of Ovine Lutropin or its Beta Subunit by Endoproteinase Arg-C:  Properties of the Arg (Β-43) cleaved Hormone.  J. Biol. Chem.,  263, 12602-12607.
15. Ward, D.N., Bousfield, G.R., Gordon, W.L., & Sugino, H. 1988. Chemistry of the Peptide Components of Glycoprotein Hormones.  In: Keel, B.A. and Grotjan, H.E., Jr., (eds).  Microheterogeneity of Glycoprotein Hormones,  Boca Raton: CRC Press, 1-21. 
16. Ward, D.N., Hines, K.K., Gordon, W.L. & Bousfield, G.R. 1988. The Purification of Native Inhibin and Chemical Characterization.  In: Hodgen, G.D., Rosenwaks, Z., and  Spieler, J.M., (eds.) Nonsteroidal Gonadal Factors: Physiological Roles and Possibilities in Contraceptive Development, Norfolk: The Jones Institute Press, 1-16.
17. Bousfield, G. R., Liu, W-K., & Ward, D. N. 1989. Effects of Removal of Carboxyterminal Extension From Equine LH Beta-Subunit.  Endocrinology, 124, 379-387.
18. Bidart, J., Troalen, F., Bousfield, G. R., Bohuon, C., & Bellet, D. 1989. Monoclonal Antibodies Directed to Human and Equine Chorionic Gonadotropins as Probes for the Topographic Analysis of Epitopes on the Human Beta-Subunit.  Endocrinology, 124, 923-929.
19. Gordon, W.L., Bousfield, G.R. & Ward, D.N. 1989. Comparative binding of FSH to chicken and rat testis.  J. Endocrinol. Invest., 12, 383-392.
20. Ward, D.N., Bousfield, G.R., & Mar, A.O. 1989. Chemical Reduction-Reoxidation of the Glycoprotein Hormone Disulfide Bonds.  In: Bellet, D. and  Bidart, J.-M., (eds.) Structure-Function Relationship of Gonadotropins, New York: Raven Press, 1-19.
21. Moore, K.H., Dunbar, B.S., Bousfield, G.R., & Ward, D.N. 1990. The Heterogeneity of  Porcine 32,000 Mr Inhibin alpha-subunit:  A Gel Electrophoresis and Immunoblot Study.  Endocrinology, 127, 1477-1486.
22. Ward, D.N., Bousfield, G.R., & Moore, K.H. 1990. Gonadotropins.  In: Cupps, P.T., (ed.) Reproduction in Domestic Animals, 4th ed., Orlando, FL: Academic  Press, 25-80.
23. Matsui, T., Sugino, H., Miura, M., Bousfield, G.R., Ward, D.N., Titani, K., & Mizouchi, T. 1991. Beta-subunits of equine chorionic gonadotropin and luteinizing hormone with an identical amino acid sequence have different asparagine-linked oligosaccharide chains.  Biochem. Biophys. Res. Commun.,  174, 940-945.
24. Mizuno M., Farach–Carson M.C., Pinero G.J., Fujisawa R., Brunn J.C., Seyer J.M., Bousfield G.R., Mark M.P., & Butler W.T. 1992. Identification of the rat bone 60K acidic glycoprotein as alpha2HS–glycoprotein. Bone Miner, 13, 1-21.
25. Bousfield, G.R. & Ward, D.N. 1992. Reduction and reoxidation of equine gonadotropin alpha–subunits. Endocrinology, 131, 2986-2998.
26. Smith P.L., Bousfield G.R., Kumar S., Fiete D., & Baenziger J.U.  1993. Equine lutropin and chorionic gonadotropin bear oligosaccharides terminating with SO4–4–GalNAc and siaα2,3Gal respectively. J. Biol. Chem., 268, 795-802.
27. Couture L., Lemonnier J.P., Troalen F., Roser J.F., Bousfield G.R., Bellet D., & Bidart J.M. 1993. Immunochemical studies of equine chorionic gonadotropin (eCG), eCG–α and eCG–Β. Endocrinology, 132, 205-211.
28. Bousfield, G.R., Perry W.M., & Ward, D.N.  1994. Gonadotropins: chemistry and biosynthesis.In: Knobil E., Neill J.D. (ed.) The Physiology of Reproduction, New York: Raven Press, 1749-1792.
29. Moore K.H., Dunbar B.S., Bousfield G.R., & Ward D.N. 1994. Initial Characterization of Equine Inhibin. Biol. Reprod., 51, 63-71.
30. Bousfield, G.R. & Ward, D.N. 1994. Evidence for Two Folding Domains in Glycoprotein Hormone Alpha-Subunits. Endocrionology, 135, 624-635.
31. Matsui, T., Mizuochi, T., Titani, K., Oinaga, T., Hoshi, M., Bousfield, G.R., Sugino, H., & Ward, D.N.  1994. Structural Analysis of N–Linked Oligosaccharides of Equine Chorionic Gonadotropin and Lutropin Beta–Subunits. Biochemistry, 33, 14039-14048.
32. Robert, P., Troalen, F., Bellet, D., Bousfield, G. R. & Bidart, J.-M. 1994. Immunochemical mapping of human lutropin: I. delineation of a conformational antigenic determinant. Mol. Cell. Endocrinol., 101, 11-20.
33. Butnev, V.Y., Gotschall, R.R., Baker, V.L., Moore, W.T., & Bousfield, G.R. 1996. Negative Influence of O–linked Oligosaccharides of High Molecular Weight Equine Chorionic Gonadotropin (eCG) on its LH and FSH Receptor Binding Activities. Endocrinology, 137, 2530-2542.
34. Gotschall, R.R. & Bousfield, G.R. 1996. Oligosaccharide mapping reveals hormone–specific glycosylation patterns on eqiune gonadotropin alpha-subunit Asn56. Endocrinology, 137, 2543-2557.
35. Butnev, V.Y., Gotschall, R.R., Baker, V.L., Moore, W.T., Gout, P.W. & Bousfield, G.R. 1996. Glycosylated equine prolactin and its carbohydrate moiety. J. Protein Chem., 15, 413-426.
36. Peng, K.C., Bousfield, G.R., Puett, D., & Ward, D.N.  1996. Circular dichroic spectroscopy of Arg46-nicked ovine lutropin α and derived fragments. J Protein Chem., 15, 547-552.
37. Bousfield GR, Butnev VY, Gotschall RR, Baker VL, Moore WT:  Structural features of mammalian gonadotropins. Mol. Cell. Endocrinol. 125: 3–19, 1996
38. Butnev, V,Y., Gotschall, R.R., Butnev, V.Y., Baker, V.L., Moore, W.T., Bousfield, G.R. 1998. Inhibitory influence of alpha-subunit Asn56 oligosaccharide on in vitro subunit association and FSH receptor binding of equine gonadotropins. Biol. Reprod., 58, 458–469.
39. Bousfield, G.R. 1999. LH (Luteinizing Hormone). In: Knobil, E. and Neill, J.D., (eds.) Encyclopedia of Reproduction, San Diego: Academic Press, Vol. 2, 1034-1054.
40. Bousfield, G.R., Baker, V.L., Gotschall, RR., Butnev, V.Y., & Butnev, V.Y. 2000. Carbohydrate Analysis of Glycoprotein Hormones. Methods, 21, 15-39.
41. Bousfield, G.R., Butnev, V.Y. & Butnev, V.Y. 2001. Identifcation of 12 O-glycosylation sites in eLH and eCG alpha subunits by solid-phase Edman degradation. Biol. Reprod., 64, 136-147.
42. Walton, W.J., Nguyen, V.T., Butnev, V.Y., Singh, V., Moore, W.T., & Bousfield, G.R. 2001. Characterization of Human Follicle-Stimulating Hormone Isoforms Reveals a Non-Glycosylated Beta-subunit In Addition to the Conventional Glycosylated Alpha-subunit. J. Clin. Endocrinol. Metab., 86, 3675-3585.
43. Butnev, V.Y., Singh, V., Nguyen, V.T., & Bousfield, G.R. 2002. Truncated eLHΒ, Asparagine56-deglycosylated-eLH alpha Combine to Produce a Potent Follicle-Stimulating Hormone Antagonist.  J. Endocrinol., 172, 545-555.
44. Bousfield, G.R. & Butnev, V.Y. 2002. Equine gonadotropins as models for glycosylation studies. Indian J. Exptl. Biology, 40, 486-506.
45. Nguyen, V.T., Singh, V., Butnev, V.Y., Gray, C.M., Westfall, S., Davis, J.S., Dias, J.A., & Bousfield, G.R. 2003. Inositol Phosphate Stimulation by LH Requires the Entire Alpha Asn56 Oligosaccharide. Molec. Cell. Endocrinol., 199, 73-86.
46. Jiang, H., Butnev, V.Y., Bousfield, G.R., & Desaire, H. 2004. Glycoprotein profiling by electrospray mass spectrometry. J. Am. Soc. Mass Spectrom., 15, 750-758.
47. Bousfield, G.R., Butnev, V.Y., Butnev, V.Y., Nguyen, V.T., Gray, C.M., Dias, J.A., MacColl, R., Eisele, L., & Harvey, D.J.,  2004. Differential Effects of Alpha Asparagine56 Oligosaccharide Structure on Equine Lutropin and Follitropin Hybrid Conformation and Receptor-Binding Activity. Biochemistry, 43, 10817-10813.
48. Harvey, D.J., & Bousfield G.R., 2005. Differentiation between sulphated and phosphated carbohydrates in low resolution MALDI mass spectra. Rapid Communications in Mass Spectrometry, 19, 287-288.
49. Bousfield, G.R., Jia, L., & Ward, D.N. 2006. Gonadotropins: Chemistry and Biosynthesis. In:Neill J.D. (ed.) Knobil and Neill: Physiology of Reproduction, 3rd edition. San Diego: Elsevier Press, 1581-1634.
50. Irungu, J.A., Dalpathado, D.S., Go, E.P., Jiang, H., Ha, H.V., Bousfield, G.R., & Desaire, H., 2006. A Method for Characterizing Sulfated Glycoproteins in a Glycosylation Site-Specific Fashion, Using Ion-Pairing and Tandem Mass Spectrometry. Anal. Chem., 78, 11801-1190.
51. Dalpathado, D.S., Irungu, J.A., Go, E.P., Butnev, V.Y., Norton, K., Bousfield, G.R., & Desaire, H. 2006. Comparative glycomics of the glycoprotein follicle-stimulating hormone (FSH): Glycopeptide analysis of isolates from two mammalian species. Biochemistry, 45, 8665-8673.
52. Bousfield, G.R., Butnev, V.Y., Walton, W.J., Nguyen, V.T., Huneidi, J., Singh, V., Kolli, K., Harvey, D.J., & Rance, N.E. 2007. All-or-None N-Glycosylation in Primate Follicle-Stimulating Hormone Beta-Subunits. Mol. Cell. Endocrinol., 260-262, 40-48.
53. Jablonka-Shariff, A., Roser, J.F., Bousfield, G.R., Wolfe, M.W., Sibley, L.E., Colgin, M., & Boime, I. 2007. Expression And Bioactivity Of A Single Chain Recombinant Equine Luteinizing Hormone (ReLH). Theriogenology,  67, 311-320.
54. Bousfield, G.R., Butnev, V.Y., Bidart, J.M., Dalpathado, D., Irungu, J., & Desaire, H. 2008. Chromatofocusing fails to separate hFSH isoforms on the basis of glycan structure.Biochemistry, 47, 1708-1720.
55. Ulloa-Aguirre, A., Dias, J.A,. & Bousfield, G.R. 2009. Gonadotropins and the importance of glycosylation. In: Walsh, G. (ed.) Posttranslational Modifications of Protein Biopharmaceuticals, Wiley-Blackwell, 109-147.
56. Wehbi, V., Tranchant, T., Durand, G., Musnier, A., Decourtye, J., Piketty, V., Butnev, V.Y., Bousfield, G.R., Crepieux, R., Maurel, M.C., & Reiter, E. 2010. Partially deglycosylated equine LH preferentially activates Beta-arrestin-dependent signaling at the follicle-stimulating hormone receptor. Mol. Endocrinol.,  24, 561-573. 
57. Visvanathan, M., Siddam, S.R., Lee, I.H., Lushington, G.H., & Bousfield, G.R.  2011. GlycomicsDB – a data integration platform for glycans and their structures. Open Med. Inform. J.,  5, 9-16. 
58. Bousfield, G.R. & Dias, J.A., 2011. Synthesis and secretion of gonadotropins including structure-function correlates. Rev. Endocr. Metab. Disord., 12(4),  289-302.
59. Ulloa-Aguirre, A., Dias, J.A., Bousfield, G.R., Huhtaniemi, I. & Reiter, E. 2013. Trafficking of the Follitropin Receptor. Meth. Enzymol., 251, 17-45.
60. Bousfield, G.R., Butnev, V.Y., Butnev, V.Y., Hiromasa, Y., Harvey, D.J., & May, J.V. 2014. Hypo-glycosylated human follicle-stimulating hormone (hFSH21/18) is much more active in vitro than fully-glycosylated hFSH (hFSH24). Mol. Cell. Endocrinol., 382, 989-997.
61. Bousfield, G. R., Butnev, V. Y., Rueda-Santos, M. A., Brown, A., Smalter Hall, A., & Harvey, D. J. 2014. Macro and micro heterogeneity in pituitary and urinary follicle-stimulating hormone glycosylation. J. Glycomics Lipidomics, 4, 125.
62. Davis, J. S., Kumar, T. R., May, J. V., & Bousfield, G. R. 2014. Naturally occurring follicle-stimulating hormone glycosylation variants. J. Glycomics Lipidomics, 4, e117.
63. Ulloa-Aguirre, A., Reiter, E., Bousfield, G., Dias, J. A., & Huhtaniemi, I. 2014. Constitutive activity in gonadotrophin receptors. Adv. Pharmacol., 70, 37-80.
64. Bousfield, G. R., Butnev, V. Y., White, W. K., Smalter Hall, A., & Harvey, D. J. 2015. Comparison of follicle-stimulating hormone glycosylation microheterogeneity by quantitative negative mode nano-electrospray mass spectrometry of peptide-N-glycanase-released oligosaccharides. J. Glycomics Lipidomics, 5(1), 129.
65. Butnev, V. Y., Butnev, V. Y., May, J. V., Shuai, B., Tran, P., White, W. K., & Bousfield, G. R. Production, purification, and characterization of recombinant hFSH glycoforms for functional studies. Mol. Cell. Endocrinol., 405, 41-52.
66. Cohen, L., Bousfield, G. R., & Ben-Menahem, D. 2015. Recombinant equine LHβ subunit combines divergent intracellular traits of the human LHβ and CGβ subunits. Theriogenology, 83(9), 1469-1476.
67. Jiang, C., Hou, X., Wang, C., May, J. V., Butnev, V. Y., Bousfield, G. R., & Davis, J. S. 2015. Hypo-glycosylated hFSH has greater bioactivity than fully-glycosylated recombinant hFSH in human granulosa cells. J. Clin. Endocrinol. Metab., 100(6), e852-E860.
68. Meher, B. R., Dixit, A., Bousfield, G. R., & Lushington, G. H. 2015. Glycosylation effects on FSH-FSHR interaction dynamics: A case study of different FSH glycoforms by molecular dynamics simulations. PLoS ONE, 10(9), e0137897.
69. Wang, H., Butnev, V. Y., Bousfield, G. R., & Kumar, T. R. 2016. A human FSHB transgene encoding the double N-glycosylation mutant (Asn^7Δ Asn^25Δ) FSHβ fails to rescue Fshb null mice. Mol. Cell. Endocrinol., 426, 113-124.
70. Wang, H., May, J., Shuai, B., May, J. V., Bousfield, G. R., & Kumar, T. R. 2016. Evaluation of in vivo bioactivities of recombiant hypo-(FSH^21/18) and fully- (FSH²⁴) glycosylated human FSH glycoforms in Fshb null mice. Mol. Cell. Endocrinol., 437, 224-236.
71. Ulloa-Aguirre, A., Dias, J. A., & Bousfield, G. R. 2017. Gonadotropins. In: Simoni, M. & Huhtaniemi, I. (Eds.) Endocrinology of the Testis and Male Reproduction Springer, Cham., 1-52.
72. Bousfield, G. R., May, J. V., Davis, J. S., Dias, J. A., & Kumar, T. R. 2018. In vivo and in vitro impact of carbohydrate variation on follicle-stimulating hormone function. Front. Endocrinol., 9, 216.
73. Bousfield, G. R. 2019. Biosynthesis and Posttranslational Processing of Peptide Hormones.Reference Module in Biomedical Sciences., 

    doi: 10.1016/B978-0-12-801238-3.92822-8.

74. Bousfield, G. R. & Harvey, D.J. 2019. Follicule-Stimulating Hormone Glycobiology.Endocrinology, 160(6), 1515-1535.

Professional Experience

Professional Experience

Awards and Honors

Professional Organizations and Honors

• Sigma Xi
• American Association for the Advancement of Science
• American Chemical Society
• American Society for Biochemistry and Molecular Biology
• The Endocrine Society
• The Protein Society
• Society for the Study of Reproduction
• Society for Glycobiology
• Association of Biomedical Resource Facilities
• 1974, William B. Owsley Outstanding Student in Biology Award, Presented by Saginaw Valley State College
• 1994 Wichita State University Mortar Board, Educator Appreciation Award
• 2003 Kansas Biomedical Research Infrastructure Network Faculty Scholar Award
• 2010 Wichita State University Excellence in Research Award

Grants

 Awarded Grants

Additional Information