- Library Home /
- Search Collections /
- Open Collections /
- Browse Collections /
- UBC Theses and Dissertations /
- Characterization of mouse CD43 recombinant proteins...
Open Collections
UBC Theses and Dissertations
UBC Theses and Dissertations
Characterization of mouse CD43 recombinant proteins secreted by EL4, CTL2c, CTLL and NSF60 cells Yang, Jeanne Chi-Mei
Abstract
Leukosialin (CD43) is a heavily glycosylated mucin-type acidic cell surface protein, carrying 70-80 O-glycans. The molecular weight heterogeneity of both human and murine CD43 glycoforms are due to the variation in their O-linked glycans. There are two major CD43 glycoforms. Resting T cells express predominantly a 115 kDa glycoform of CD43, carrying mainly tetrasaccharide side chains, whereas activated T cells carry mainly hexasaccharide side chains and express CD43 as a 135 kDa glycoform. The activity of (31- 6GlcNAc transferase (C2GnT) has been shown to be correlated with the expression of CD43 135 kDa. Since the function of the two major CD43 glycoforms is not clear, CD43 chimeric proteins have been produced to identify potential ligands of CD43 and to study its role(s) in the immune response. The recombinant chimeric glycoprotein comprises the extracellular domain of murine CD43 (mCD43) and part of human IgGl (hlgG) including the hinge, CH2 and CH3 domains. The cDNA encoding the mCD43-hIgG chimeric molecule was subcloned into two expression vectors which are driven by either metallothionein or SRa promoter. Both vectors were transfected into three T cell lines: EL4, CTL2c, CTLL and myeloid cell line, NSF60. EL4 cells express exclusively CD43 115 kDa which is specifically recognized by the monoclonal antibody (mAb), S7 whereas CTL2c and CTLL cells express exclusively CD43 135 kDa which is specifically recognized by mAb 1B11. NSF60 cells express CD43 glycoforms which are detected by S7 and IB 11. Western blotting analysis demonstrated that the anti-CD43 antibody reactivity of the chimeras secreted by transfected EL4, CTL2c, CTLL and NSF60 cells was identical to the cell surface CD43. The EL4 chimera was recognized by mAb S7, while the CTL2c and CTLL chimeras were recognized by mAb 1B11. NSF60 cells secreted a chimera which was reactive with both antibodies. These results suggest that the mCD43-hIgG chimera secreted by transfected EL4 cells predominantly carried tetrasaccharide cores whereas the chimeras secreted by transfected CTL2c and CTLL cells predominantly carried hexasaccharide cores. NSF60 chimeric protein had tetrasaccharide and hexasaccharide structures. The four chimeras had a lOkDa higher MW than CD43 expressed on the surface of its corresponding cells. This 10 kDa difference in MW can be fully explained by the replacement of the transmembrane and cytoplasmic domains of CD43 with pools of the human IgG constant domain. This further indicated that chimeras have similar or identical glycosylation as cell surface CD43. Non-reducing SDS-PAGE showed that all four chimeras were as expected secreted as dimers. Precipitation using Jacalin-sepharose specific for tetrasaccharide cores, demonstrated that CD43 115 kDa expressed on EL4 cells and its corresponding 125 kDa chimera were efficiently precipitated, while the other three chimeric proteins were less reactive with Jacalin sepharose. This result supported that the O-glycans expressed on the chimeric proteins were identical to those expressed on CD43. This study has successfully established novel tools for the search of CD43 glycoforms specific ligands.
Item Metadata
Title |
Characterization of mouse CD43 recombinant proteins secreted by EL4, CTL2c, CTLL and NSF60 cells
|
Creator | |
Publisher |
University of British Columbia
|
Date Issued |
1996
|
Description |
Leukosialin (CD43) is a heavily glycosylated mucin-type acidic cell surface
protein, carrying 70-80 O-glycans. The molecular weight heterogeneity of both human and
murine CD43 glycoforms are due to the variation in their O-linked glycans. There are two
major CD43 glycoforms. Resting T cells express predominantly a 115 kDa glycoform of
CD43, carrying mainly tetrasaccharide side chains, whereas activated T cells carry mainly
hexasaccharide side chains and express CD43 as a 135 kDa glycoform. The activity of (31-
6GlcNAc transferase (C2GnT) has been shown to be correlated with the expression of CD43
135 kDa. Since the function of the two major CD43 glycoforms is not clear, CD43 chimeric
proteins have been produced to identify potential ligands of CD43 and to study its role(s) in the
immune response.
The recombinant chimeric glycoprotein comprises the extracellular domain of
murine CD43 (mCD43) and part of human IgGl (hlgG) including the hinge, CH2 and CH3
domains. The cDNA encoding the mCD43-hIgG chimeric molecule was subcloned into two
expression vectors which are driven by either metallothionein or SRa promoter. Both vectors
were transfected into three T cell lines: EL4, CTL2c, CTLL and myeloid cell line, NSF60.
EL4 cells express exclusively CD43 115 kDa which is specifically recognized by the
monoclonal antibody (mAb), S7 whereas CTL2c and CTLL cells express exclusively CD43
135 kDa which is specifically recognized by mAb 1B11. NSF60 cells express CD43
glycoforms which are detected by S7 and IB 11. Western blotting analysis demonstrated that
the anti-CD43 antibody reactivity of the chimeras secreted by transfected EL4, CTL2c, CTLL
and NSF60 cells was identical to the cell surface CD43. The EL4 chimera was recognized by
mAb S7, while the CTL2c and CTLL chimeras were recognized by mAb 1B11. NSF60 cells
secreted a chimera which was reactive with both antibodies. These results suggest that the
mCD43-hIgG chimera secreted by transfected EL4 cells predominantly carried tetrasaccharide
cores whereas the chimeras secreted by transfected CTL2c and CTLL cells predominantly
carried hexasaccharide cores. NSF60 chimeric protein had tetrasaccharide and hexasaccharide
structures. The four chimeras had a lOkDa higher MW than CD43 expressed on the surface of
its corresponding cells. This 10 kDa difference in MW can be fully explained by the replacement
of the transmembrane and cytoplasmic domains of CD43 with pools of the human IgG
constant domain. This further indicated that chimeras have similar or identical glycosylation as
cell surface CD43.
Non-reducing SDS-PAGE showed that all four chimeras were as expected
secreted as dimers. Precipitation using Jacalin-sepharose specific for tetrasaccharide cores,
demonstrated that CD43 115 kDa expressed on EL4 cells and its corresponding 125 kDa
chimera were efficiently precipitated, while the other three chimeric proteins were less reactive
with Jacalin sepharose. This result supported that the O-glycans expressed on the chimeric
proteins were identical to those expressed on CD43. This study has successfully established
novel tools for the search of CD43 glycoforms specific ligands.
|
Extent |
9961962 bytes
|
Genre | |
Type | |
File Format |
application/pdf
|
Language |
eng
|
Date Available |
2009-03-09
|
Provider |
Vancouver : University of British Columbia Library
|
Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
|
DOI |
10.14288/1.0087606
|
URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
|
Graduation Date |
1997-05
|
Campus | |
Scholarly Level |
Graduate
|
Aggregated Source Repository |
DSpace
|
Item Media
Item Citations and Data
Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.