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Carbohydrate groups of alpha1-microglobulin are important for secretion and tissue localization but not for immunological properties

Wester Rosenlöf, Lena LU ; Fast, Jonas LU ; Labuda, T; Cedervall, Tommy LU ; Wingårdh, Karin LU ; Olofsson, Tor LU and Åkerström, Bo LU (2000) In Glycobiology 10(9). p.891-900
Abstract
The role of the carbohydrates for the structure and functions of the plasma and tissue protein alpha1-microglobulin (alpha1m) was investigated by deletion of the sites for N-glycosylation by site-directed mutagenesis (N17,96-->Q). The mutated cDNA was expressed in a baculovirus-insect cell system resulting in a nonglycosylated protein. The biochemical properties of N17,96Q-alpha1m were compared to nonmutated alpha1m, which carries two short non-sialylated N-linked oligosaccharides when expressed in the same system. Both proteins carried a yellow-brown chromophore and were heterogeneous in charge. Circular dichroism spectra and antibody binding indicated a similar overall structure. However, the secretion of N17,96Q-alpha1m was... (More)
The role of the carbohydrates for the structure and functions of the plasma and tissue protein alpha1-microglobulin (alpha1m) was investigated by deletion of the sites for N-glycosylation by site-directed mutagenesis (N17,96-->Q). The mutated cDNA was expressed in a baculovirus-insect cell system resulting in a nonglycosylated protein. The biochemical properties of N17,96Q-alpha1m were compared to nonmutated alpha1m, which carries two short non-sialylated N-linked oligosaccharides when expressed in the same system. Both proteins carried a yellow-brown chromophore and were heterogeneous in charge. Circular dichroism spectra and antibody binding indicated a similar overall structure. However, the secretion of N17,96Q-alpha1m was significantly reduced and approximately 75% of the protein were found accumulated intracellularly. The in vitro immunological effects of recombinant nonmutated alpha1m and N17,96Q-alpha1m were compared to the effects of alpha1m isolated from plasma, which is sialylated and carries an additional O-linked oligosaccharide. All three alpha1m variants bound to human peripheral lymphocytes and mouse T cell hybridomas to the same extent. They also inhibited the antigen-stimulated proliferation of peripheral lymphocytes and antigen-stimulated interleukin 2-secretion of T cell hybridomas in a similar manner. After injection of rats intravenously, the blood clearance of recombinant nonmutated and N17,96Q-alpha1m was faster than that of plasma alpha1m. Nonmutated alpha1m was located primarily to the liver, most likely via binding to asialoglycoprotein receptors, and N17,96Q-alpha1m was located mainly to the kidneys. It is concluded that the carbohydrates of alpha1m are important for the secretion and the in vivo turnover of the protein, but not for the structure or immunological properties. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Glycobiology
volume
10
issue
9
pages
891 - 900
publisher
Oxford University Press
external identifiers
  • pmid:10988251
ISSN
1460-2423
DOI
10.1093/glycob/10.9.891
language
English
LU publication?
yes
id
1198f64c-99e6-4f04-99fc-cd909ae4a791 (old id 1117989)
date added to LUP
2008-06-19 14:25:00
date last changed
2016-04-16 02:57:13
@article{1198f64c-99e6-4f04-99fc-cd909ae4a791,
  abstract     = {The role of the carbohydrates for the structure and functions of the plasma and tissue protein alpha1-microglobulin (alpha1m) was investigated by deletion of the sites for N-glycosylation by site-directed mutagenesis (N17,96-->Q). The mutated cDNA was expressed in a baculovirus-insect cell system resulting in a nonglycosylated protein. The biochemical properties of N17,96Q-alpha1m were compared to nonmutated alpha1m, which carries two short non-sialylated N-linked oligosaccharides when expressed in the same system. Both proteins carried a yellow-brown chromophore and were heterogeneous in charge. Circular dichroism spectra and antibody binding indicated a similar overall structure. However, the secretion of N17,96Q-alpha1m was significantly reduced and approximately 75% of the protein were found accumulated intracellularly. The in vitro immunological effects of recombinant nonmutated alpha1m and N17,96Q-alpha1m were compared to the effects of alpha1m isolated from plasma, which is sialylated and carries an additional O-linked oligosaccharide. All three alpha1m variants bound to human peripheral lymphocytes and mouse T cell hybridomas to the same extent. They also inhibited the antigen-stimulated proliferation of peripheral lymphocytes and antigen-stimulated interleukin 2-secretion of T cell hybridomas in a similar manner. After injection of rats intravenously, the blood clearance of recombinant nonmutated and N17,96Q-alpha1m was faster than that of plasma alpha1m. Nonmutated alpha1m was located primarily to the liver, most likely via binding to asialoglycoprotein receptors, and N17,96Q-alpha1m was located mainly to the kidneys. It is concluded that the carbohydrates of alpha1m are important for the secretion and the in vivo turnover of the protein, but not for the structure or immunological properties.},
  author       = {Wester Rosenlöf, Lena and Fast, Jonas and Labuda, T and Cedervall, Tommy and Wingårdh, Karin and Olofsson, Tor and Åkerström, Bo},
  issn         = {1460-2423},
  language     = {eng},
  number       = {9},
  pages        = {891--900},
  publisher    = {Oxford University Press},
  series       = {Glycobiology},
  title        = {Carbohydrate groups of alpha1-microglobulin are important for secretion and tissue localization but not for immunological properties},
  url          = {http://dx.doi.org/10.1093/glycob/10.9.891},
  volume       = {10},
  year         = {2000},
}