Molecular weights of CTLA-4 and CD80 by sedimentation equilibrium ultracentrifugation.
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 Th-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 Th-cells and antigen-presenting cells.
Fairman
R
R
Department of Biology, Haverford College, 370 Lancaster Avenue, Haverford, Pennsylvania 19041, USA. rfairman@haverford.edu
Fenderson
W
W
Hail
M E
ME
Wu
Y
Y
Shaw
S Y
SY
D000069594:Abatacept; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000737:chemistry*; D018122:B7-1 Antigen / Q000737:chemistry*; D060908:CTLA-4 Antigen; D002240:Carbohydrate Sequence; D002499:Centrifugation, Density Gradient / Q000379:methods*; D019281:Dimerization; D018796:Immunoconjugates; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation
D015703:Antigens, CD; D000943:Antigens, Differentiation; D018122:B7-1 Antigen; D060908:CTLA-4 Antigen; D018796:Immunoconjugates; D000069594:Abatacept