TY - JOUR
T1 - Molecular weights of CTLA-4 and CD80 by sedimentation equilibrium ultracentrifugation
AU - Fairman, Robert
AU - Fenderson, William
AU - Hail, Mark E.
AU - Wu, Youling
AU - Shaw, Shyh Yu
PY - 1999/6/1
Y1 - 1999/6/1
N2 - Proteins that are heavily glycosylated pose unique challenges in their biophysical characterization. In particular, molecular weight analysis is exacerbated by such glycosylation. For example, glyoproteins are refractory to careful mass spectrum analysis and often give anomalous retention times using size exclusion chromatography. We combine several approaches to characterize the molecular weights of the extracellular domains of the glycoproteins CTLA-4 and CD80 using carbohydrate analysis, electrospray mass spectrometry, size exclusion chromatography, and analytical ultracentrifugation. In addition, we have applied a method described previously, using sedimentation equilibrium analysis to calculate the contribution of carbohydrates to the molecular masses of CTLA-4 and CD80. It is important to understand the oligomeric states of these protein domains because the interaction between these lymphocyte receptors plays an important costimulatory role in the T(h)-cell antigenic response. It is thought that extracellular interactions between these receptors may regulate both the self-association of these receptor proteins and the oligomeric state of the heterocomplex; this regulation has important consequences for potentiating the signaling mechanism between T(h)-cells and antigen-presenting cells.
AB - Proteins that are heavily glycosylated pose unique challenges in their biophysical characterization. In particular, molecular weight analysis is exacerbated by such glycosylation. For example, glyoproteins are refractory to careful mass spectrum analysis and often give anomalous retention times using size exclusion chromatography. We combine several approaches to characterize the molecular weights of the extracellular domains of the glycoproteins CTLA-4 and CD80 using carbohydrate analysis, electrospray mass spectrometry, size exclusion chromatography, and analytical ultracentrifugation. In addition, we have applied a method described previously, using sedimentation equilibrium analysis to calculate the contribution of carbohydrates to the molecular masses of CTLA-4 and CD80. It is important to understand the oligomeric states of these protein domains because the interaction between these lymphocyte receptors plays an important costimulatory role in the T(h)-cell antigenic response. It is thought that extracellular interactions between these receptors may regulate both the self-association of these receptor proteins and the oligomeric state of the heterocomplex; this regulation has important consequences for potentiating the signaling mechanism between T(h)-cells and antigen-presenting cells.
UR - http://www.scopus.com/inward/record.url?scp=0033152466&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0033152466&partnerID=8YFLogxK
U2 - 10.1006/abio.1999.4095
DO - 10.1006/abio.1999.4095
M3 - Article
C2 - 10334846
AN - SCOPUS:0033152466
SN - 0003-2697
VL - 270
SP - 286
EP - 295
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -