Title : Identification and mutational analysis of the glycosylation
sites of human
keratin 18
Abstract :
- Keratin polypeptides 8 and 18 (K8/18 ) are intermediate filament phosphoglycoproteins that are expressed preferentially in glandular epithelia
- We previously showed that K8/18 phosphorylation occurs on serine residues and that K8/18 glycosylation consists of O-linked single N-acetylglucosamines (O-GlcNAc) that are linked to Ser/Thr
- Since the function of these modifications is unknown, we sought as a first step to identify the precise modification sites and asked if they play a role in keratin filament assembly
- For this, we generated a panel of K18 Ser and Thr--> Ala mutants at potential glycosylation sites followed by expression in a baculovirus-insect cell system
- We identified the major glycosylation sites of K18 by comparing the tryptic 3H-glycopeptide pattern of the panel of mutant and wild type K18 expressed in the insect cells with the glycopeptides of K18 in human colonic cells
- The identified sites occur on three serines in the head domain of K18
- The precise modified residues in human cells were verified using Edman degradation and confirmed further by the lack of glycosylation of a K18 construct that was mutated at the molecularly identified sites then transfected into NIH-3T3 cells
- Partial or total K18 glycosylation mutants transfected into mammalian cells manifested nondistinguishable filament assembly to cells transfected with wild type K8/18
- Our results show that K18 glycosylation sites share some features with other already identified O-GlcNAc sites and may together help predict glycosylation sites of other intermediate filament proteins
Output (sent_index, trigger,
protein,
sugar,
site):
- 0. glycosylation, , keratin 18, -, sites
- 1. phosphoglycoproteins, , phosphoglycoproteins, -, polypeptides
- 2. linked, , -, O-GlcNAc, Ser/Thr
- 4. glycosylation, , -, -, sites
- 5. 3H-glycopeptide, , -, -, 3H-glycopeptide
- 5. glycopeptides, , K18, -, glycopeptides
- 5. glycosylation, , K18, -, sites
- 9. glycosylation, , -, -, sites
- 9. glycosylation, , proteins, -, sites
Output(Part-Of) (sent_index,
protein,
site):
- 0. keratin 18, sites
- 1. Keratin, polypeptides
- 4. K18, Ser and Thr
- 5. K18, glycopeptides
- 5. K18, sites
- 6. K18, domain
- 6. K18, serines
- 9. K18, sites
- 9. proteins, sites
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):