Stimulation of peripheral blood mononuclear cells with lipopolysaccharide induces expression of the plasma protein alpha2-macroglobulin.
The human alpha(2)-macroglobulin gene is approximately 48 kb in size and consists of 36 exons, which encode the 180 kDa subunit of this large tetrameric protein. In this investigation, a procedure of sequencing human alpha(2)-macroglobulin mRNA, using mRNA from lipopolysaccharide-stimulated peripheral blood mononuclear cells as template in RT-PCR, was developed. Incubation of peripheral blood mononuclear cell populations with lipopolysaccharide induced alpha(2)-macroglobulin mRNA expression reaching levels detectable by RT-PCR. Extracted human alpha(2)-macroglobulin mRNA was used to determine the nucleotide sequence of a 500 bp DNA segment encoding the most C-terminal, receptor-binding part of the protein, using alpha(2)-macroglobulin specific primers. The sequence obtained matched the earlier published sequence of human alpha(2)-macroglobulin, except for three point mutations, i.e., cytosine for guanine, cytosine for thymidine and thymidine for adenine substitutions at positions 4369, 4423, and 4511, respectively. None of these alterations, however, affect the amino acid sequence of the protein. In conclusion, we demonstrate a new, improved, approach to sequence human alpha(2)-macroglobulin mRNA by overexpressing the protein in peripheral blood mononuclear cells. This procedure may be useful in the search for mutations in alpha(2)-macroglobulin, examining its role in the pathogenesis of human diseases.
Gunnarsson
Martin
M
Department of Immunology, Umeå University, S-901 85 Umeå, Sweden. martin.c.gunnarsson@vll.se
Frängsmyr
Lars
L
Stigbrand
Torgny
T
Jensen
Poul Erik H
PE
D001483:Base Sequence; D003001:Cloning, Molecular; D005434:Flow Cytometry; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA; D012689:Sequence Homology, Nucleic Acid; D000511:alpha-Macroglobulins / Q000096:biosynthesis*
D008070:Lipopolysaccharides; D012333:RNA, Messenger; D000511:alpha-Macroglobulins