Expression of a cDNA encoding the glucose trimming enzyme glucosidase II in CHO cells and molecular characterization of the enzyme deficiency in a mutant mouse lymphoma cell line
| Autor: | Martin Ziak, D. Brada, Jürgen Roth, Thomas Flura |
|---|---|
| Přispěvatelé: | University of Zurich, Roth, Jürgen |
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
DNA
Complementary 1303 Biochemistry Lymphoma Transcription Genetic Swine Molecular Sequence Data 610 Medicine & health CHO Cells Biology Transfection Biochemistry Polymerase Chain Reaction 142-005 142-005 03 medical and health sciences Mice Open Reading Frames Complementary DNA Cricetinae Tumor Cells Cultured Animals Humans Amino Acid Sequence Cloning Molecular 030304 developmental biology chemistry.chemical_classification 0303 health sciences Binding Sites Base Sequence Sequence Homology Amino Acid cDNA library Chinese hamster ovary cell 030302 biochemistry & molecular biology ER retention alpha-Glucosidases Molecular biology Peptide Fragments Recombinant Proteins Amino acid Rats Open reading frame Transmembrane domain Enzyme chemistry Liver Sequence Alignment |
| Popis: | Glucosidase II is an ER resident glycoprotein involved in the processing of N-linked glycans and probably a component of the ER quality control of glycoproteins. For cloning of glucosidase II cDNA, degenerate oligonucleotides based on amino acid sequences derived from proteolytic fragments of purified pig liver glucosidase II were used. An unamplified cDNA library from pig liver was screened with a 760 bp glucosidase II specific cDNA fragment obtained by RT-PCR. A 3.9 kb glucosidase II cDNA with an open reading frame of about 2.9 kb was obtained. The glucosidase II sequence did not contain known ER retention signals nor hydrophobic regions which could represent a transmembrane domain; however, it contained a single N-glycosylation site close to the amino terminus. All studied pig and rat tissues exhibited an mRNA of approximately 4.4 kb with varying tissue expression levels. The authenticity of the identified cDNA with that coding for glucosidase II was proven by overexpressio n in CHO cells. Mouse lymphoma PHAR 2.7 cells, deficient in glucosidase II activity, were shown to be devoid of transcripts. |
| Databáze: | OpenAIRE |
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