Expression, glycosylation, and phosphorylation of human keratins 8 and 18 in insect cells

Nam-on Ku, M. Bishr Omary

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

The filament forming ability and post-translational modifications of the human intermediate filaments, keratin polypeptides 8 and 18 (K8/18), were studied in recombinant baculovirus-infected insect (Spodoptera frugiperda, Sf9) cells. No change in cell morphology was noted after high levels of K8/18 were expressed in Sf9 cells coinfected with recombinant virus-containing human K8 and K18. Immunofluorescence staining showed that K8/18 expressed in Sf9 cells formed somewhat disorganized and rope-like filaments, in contrast with K8 or K18 expression alone, which did not result in filament formation. K8/18 expressed in Sf9 cells were glycosylated (O-linked N-acetylglucosamine) and phosphorylated, and each modification occurred on different molecules of K8 and K18, as previously found in human HT29 cells. The glycosylation and phosphorylation of K18 in human and insect cells were very similar as determined by tryptic peptide mapping and localization to the head and proximal rod domains. In contrast, differences were noted in the relative intensity of the tryptic phospho- and glycopeptides of K8 expressed in human and insect cells and in the ratio of K8 to K18 phosphorylation in human and insect cells. Our results show that although quantitative differences exist, the post-translational modification of K8/18 expressed in insect cells is quite similar to its mammalian counterpart, especially for K18. Baculovirus expressed K8/18 should prove useful for mapping phosphorylation and glycosylation sites and for studying factors involved in organized filament assembly in mammalian cells.

Original languageEnglish
Pages (from-to)24-35
Number of pages12
JournalExperimental Cell Research
Volume211
Issue number1
DOIs
Publication statusPublished - 1994 Jan 1

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Keratin-18
Glycosylation
Insects
Sf9 Cells
Phosphorylation
Baculoviridae
Post Translational Protein Processing
Keratin-8
Spodoptera
Phosphopeptides
HT29 Cells
Peptide Mapping
Acetylglucosamine
Intermediate Filaments
Glycopeptides
Fluorescent Antibody Technique
K-18 conjugate
human KRT8 protein
Head
Staining and Labeling

All Science Journal Classification (ASJC) codes

  • Cell Biology

Cite this

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title = "Expression, glycosylation, and phosphorylation of human keratins 8 and 18 in insect cells",
abstract = "The filament forming ability and post-translational modifications of the human intermediate filaments, keratin polypeptides 8 and 18 (K8/18), were studied in recombinant baculovirus-infected insect (Spodoptera frugiperda, Sf9) cells. No change in cell morphology was noted after high levels of K8/18 were expressed in Sf9 cells coinfected with recombinant virus-containing human K8 and K18. Immunofluorescence staining showed that K8/18 expressed in Sf9 cells formed somewhat disorganized and rope-like filaments, in contrast with K8 or K18 expression alone, which did not result in filament formation. K8/18 expressed in Sf9 cells were glycosylated (O-linked N-acetylglucosamine) and phosphorylated, and each modification occurred on different molecules of K8 and K18, as previously found in human HT29 cells. The glycosylation and phosphorylation of K18 in human and insect cells were very similar as determined by tryptic peptide mapping and localization to the head and proximal rod domains. In contrast, differences were noted in the relative intensity of the tryptic phospho- and glycopeptides of K8 expressed in human and insect cells and in the ratio of K8 to K18 phosphorylation in human and insect cells. Our results show that although quantitative differences exist, the post-translational modification of K8/18 expressed in insect cells is quite similar to its mammalian counterpart, especially for K18. Baculovirus expressed K8/18 should prove useful for mapping phosphorylation and glycosylation sites and for studying factors involved in organized filament assembly in mammalian cells.",
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Expression, glycosylation, and phosphorylation of human keratins 8 and 18 in insect cells. / Ku, Nam-on; Omary, M. Bishr.

In: Experimental Cell Research, Vol. 211, No. 1, 01.01.1994, p. 24-35.

Research output: Contribution to journalArticle

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