O‐Glycosylated 24 kDa human growth hormone has a mucin‐like biantennary disialylated tetrasaccharide attached at Thr‐60 |
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Authors: | Juan J Bustamante Leticia Gonzalez Christopher A Carroll Susan T Weintraub Roberto M Aguilar Jesus Muñoz Andrew O Martinez Luis S Haro |
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Institution: | 1. Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, TX, USA;2. Department of Integrative Physiology, The University of North Texas Health Science Center at Fort Worth, Fort Worth, TX, USA;3. Department of Biochemistry, The University of Texas Health Science Center at San Antonio, San Antonio, TX, USA;4. Reeve‐Irvine Research Center, University of California, Irvine, CA, USA;5. Department of Chemistry, The University of Texas at San Antonio, San Antonio, TX, USA;6. Department of Biology, The University of Texas at San Antonio, San Antonio, TX, USA |
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Abstract: | MS was used to characterize the 24 kDa human growth hormone (hGH) glycoprotein isoform and determine the locus of O‐linked oligosaccharide attachment, the oligosaccharide branching topology, and the monosaccharide sequence. MALDI‐TOF/MS and ESI‐MS/MS analyses of glycosylated 24 kDa hGH tryptic peptides showed that this hGH isoform is a product of the hGH normal gene. Analysis of the glycoprotein hydrolysate by high‐performance anion‐exchange chromatography with pulsed amperometric detection and HPLC with fluorescent detection for N‐acetyl neuraminic acid (NeuAc) yielded the oligosaccharide composition (NeuAc2, N‐acetyl galactosamine1, Gal1). After β‐elimination to release the oligosaccharide from glycosylated 24 kDa hGH, collision‐induced dissociation of tryptic glycopeptide T6 indicated that there had been an O‐linked oligosaccharide attached to Thr‐60. The sequence and branching structure of the oligosaccharide were determined by ESI‐MS/MS analysis of tryptic glycopeptide T6. The mucin‐like O‐oligosaccharide sequence linked to Thr‐60 begins with N‐acetyl galactosamine and branches in a bifurcated topology with one appendage consisting of galactose followed by NeuAc and the other consisting of a single NeuAc. The oligosaccharide moiety lies in the high‐affinity binding site 1 structural epitope of hGH that interfaces with both the growth hormone and the prolactin receptors and is predicted to sterically affect receptor interactions and alter the biological actions of hGH. |
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Keywords: | Glycosylated Human growth hormone MS O‐linked oligosaccharide Structural analysis |
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