O-Glycosylated 24 kDa human growth hormone has a mucin-like biantennary disialylated tetrasaccharide attached at Thr-60.
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 (NeuAc(2), N-acetyl galactosamine(1), Gal(1)). After beta-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.
Bustamante
Juan J
JJ
Irma Lerma Rangel College of Pharmacy, Texas A&M Health Science Center, Kingsville, TX, USA.
Gonzalez
Leticia
L
Carroll
Christopher A
CA
Weintraub
Susan T
ST
Aguilar
Roberto M
RM
Muñoz
Jesus
J
Martinez
Andrew O
AO
Haro
Luis S
LS
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D006031:Glycosylation; D019382:Human Growth Hormone / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009077:Mucins / Q000737:chemistry*; D009844:Oligosaccharides / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013912:Threonine / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism
D009077:Mucins; D009844:Oligosaccharides; D019382:Human Growth Hormone; D013912:Threonine; D014357:Trypsin