Purification and characterization of the soluble interleukin-6 receptor from human plasma and identification of an isoform generated through alternative splicing.
The soluble human interleukin-6 receptor (shIL6R) was purified from human plasma. In a single immunoaffinity purification step a 140000-fold enrichment with a yield of 95% was achieved. A subsequent IL-6 affinity chromatography resulted in a homogeneous receptor preparation but only in a yield of less than 5%. The biological activity of the soluble receptor was clearly demonstrated by its ability to induce the synthesis of the acute-phase protein 1-antichymotrypsin in HepG2 cells stably transfected with IL-6. Upon gel filtration, the native shIL6R showed an apparent molecular mass of 93 kDa. Analysis by SDS/PAGE revealed an apparent molecular mass of 65 kDa for the soluble receptor. Deglycosylation with peptide N-glycosidase F led to a shift in molecular mass from 65 kDa to 45 kDa. It has previously been shown that the shIL6R can be generated by shedding the membrane-bound form or by expression of an alternatively spliced mRNA. Here we show that the shIL6R isolated from human plasma is recognized by an affinity-purified peptide antibody raised against an amino acid sequence unique for the alternatively spliced isoform. Thus, the shIL6R isoform generated through alternative splicing which has been previously detected in supernatants of cultured cell lines is also an in vivo product circulating in human plasma.
Müller-Newen
G
G
Institut für Biochemie der Rheinisch-Westfälischen Technischen Hochschule Aachen, Germany.
Köhne
C
C
Keul
R
R
Hemmann
U
U
Müller-Esterl
W
W
Wijdenes
J
J
Brakenhoff
J P
JP
Hart
M H
MH
Heinrich
P C
PC
D017398:Alternative Splicing; D000581:Amidohydrolases; D015703:Antigens, CD / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis / Q000378:metabolism; D008970:Molecular Weight; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D012333:RNA, Messenger / Q000378:metabolism; D018123:Receptors, Interleukin / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D015850:Interleukin-6; D012333:RNA, Messenger; D018123:Receptors, Interleukin; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase