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1.
Article in Chinese | WPRIM | ID: wpr-562459

ABSTRACT

Objective The C-terminal domain of rodent Muc3 is proteolytically cleaved.This study is to explore the relationship between N-linked oligosaccharides in SEA module and the proteolytic cleavage within C-terminal domain of rodent Muc3.Methods Truncated rodent Muc3 C-terminal domains with complete SEA module(p20SEA) were produced by site-directed mutagenesis to insert a stop code in the required place.Proteins were detected by pulse/chase and immunoprecipitation method,or SDS/PAGE and Western blot.Inhibition of glycosylation of the expressed protein was performed by using tunicamycin.Results Muc3 C-terminal domain was posttranslationally cleaved to produce a V5-tagged 30 000 extracellular glycopeptide and a Myc-tagged 49 000 membrane-associated glycopeptide.Treatment with tunicamycin to transfected COS-1 cells led to the abundant production of 60 000 uncleaved and whole-length Muc3 C-terminal domain,the 30 000 N-terminal fragment shifted to 22 000 and 49 000 C-terminal fragment shifted to 41 000 after deglycosylation.The truncated Muc3 C-terminal domain containing complete SEA module but without the following residues led to production of 36 000 uncleaved and whole-length protein,and 30 000 cleaved product shifted to 22 000 after deglycosylation.Conclusion Proteolytic cleavage in both complete rodent C-terminal domain and complete SEA module without the following residues were partially inhibited by tunicamycin.

2.
Article in Chinese | WPRIM | ID: wpr-563837

ABSTRACT

Objective To explore the correlation between membrane targeting of rodent Muc3 C-terminal domain and proteolytic cleavage blockage within its SEA module and N-linked oligosaccharides inhibition.Methods COS-1 cells were transfected with three different expression vectors containing rodent Muc3 C-terminal domain,namely p20,p20t and p20s/a by lipofectAMINE reagent.Inhibition of N-glycosylation of the expressed protein was performed by using tunicamycin.The transfected COS-1 cells(fixed or unfixed) were detected by immunolocalization experiments(anti-V5 and anti-Myc antibody) for the protein expression.Results In fixed COS-1 cells,the expressed product of p20 transfectant detected using both anti-Myc and anti-V5 antibodies was found to localize in perinuclear position and on the plasma membrane.While in the unfixed cells,immunostaining was only confined on cell surface using anti-V5 antibody.The expressed product of p20t transfectant was detected by anti-V5 antibody to localize only in perinuclear region,as observed in a few fixed cells.The distribution of p20s/a fluorescence resembled that of p20 transfectant.Plasma membrane targeting of the non-glycosylated products due to tunicamycin treatment still occurred in transfected COS-1 cells and resembled the glycosylated products.Conclusions The blockage of proteolytic cleavage within C-terminal domain of rodent Muc3 and its inhibition of N-linked oligosaccharides in SEA module cannot affect its membrane targeting.The only apparent requirement for membrane targeting is the transmembrane and/or cytoplasmic tail segments which exist in the C-terminal domains of rMuc3.

3.
Article in Chinese | WPRIM | ID: wpr-561760

ABSTRACT

Objective It has been known that the C-terminal domain of rodent Muc3 underwent proteolytic digestion.G/S within the motif of cleavage,LSKGSIVV,was one of the important pivots for digestion.The present investigation was aiming at exploring the unknown relationship between the integrity of SEA module and the proteolytic digestion.Method Truncated rodent Muc3 C-terminal domains(p20t and p20SEA) were produced by site-directed mutagenesis to insert a stop code in the required place.Proteins were detected by SDS/PAGE and Western blotting.Deglycosylation of the expressed protein was performed by digestion using N-glycosidase F.Results Muc3 C-terminal domain was posttranslationally cleaved to produce a V5-tagged 30kDa extracellular glycopeptide and a Myc-tagged 49kDa membrane-associated glycopeptide.The 30kDa N-terminal fragment shifted to 22kDa after deglycosylation.The truncated rodent Muc3 C-terminal domain containing complete SEA module,but without the following residues after SEA module,was 30kDa Mw as detected with anti-V5 antibody,and it was shifted to 22kDa after deglycosylation.But the truncated rodent Muc3 C-terminal domain containing incomplete SEA module(p20t) of 26-30kDa Mw was shifted to 26kDa after deglycosylation.Conclusion There was proteolytic digestion in both complete rodent C-terminal domain and complete SEA module without residues after SEA module.But proteolytic digestion does not occur in the incomplete SEA module of rodent Muc3.So it may be concluded that the integrity of SEA module of rodent Muc3 was also a crucial condition for its proteolytic digestion.

4.
J Biosci ; 1985 Sept; 9(1&2): 99-107
Article in English | IMSEAR | ID: sea-160483

ABSTRACT

Cupric complex of isonicotinic acid hydrazide inhibits DNA synthesis by avian myloblastosis virus reverse transcriptase. This inhibition occurs in the presence of either ribonucleotide or deoxyribonucleotide templates. The inhibition of reverse transcriptase by cupric-INH complex is considerably reduced when stored or proteolytically cleaved enzyme was used in the reaction. The complex also inhibits the reverse transciptase-associated RNase Η activity. The cupric-isonicotinic acid hydrazide complex cleaves pBR 322 from I DNA into smaller molecules in the presence or absence of reverse transcriptase-associated endonuclease. However, in the presence of the enzyme the DNA is cleaved to a greater extent.

5.
Article in Chinese | WPRIM | ID: wpr-558283

ABSTRACT

Objective To explore the existing molecular pattern of rodent Muc3 carboxyl-terminal domain. Methods Muc3 carboxyl-terminal domain was expressed by a transient expression system and detected with SDS/PAGE and Western blotting. Identification of interaction between two proteolytically cleaved fragments was carried out by using both metabolic labeling and immunoprecipitation of expressed proteins and affinity purification of His-tagged proteins. Results Experiments with heterologous cells transfected with cDNA encoding the 381-residue C-terminal domain of rodent Muc3 showed that a definitive proteolytic cleavage occurred during the process in the endoplasmic reticulum. The products consisted of a V5-tagged 30 000 extracellular peptide, a Myc-tagged 49 000 membrane-associated peptide and non-cleaved 55 000 of whole-length protein. Two fragments remained associated by non-covalent SDS-sensitive interactions. Conclusion The proteolytic cleavage may be a prelude to later release of the large extracellular domains at cell surfaces. But the interaction between two cleaved fragments may be an important factor to interfere with the later release of the extracellular domain.

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