Molecular Weights of CTLA-4 and CD80 by Sedimentation Equilibrium Ultracentrifugation

Fairman, Robert; Fenderson, William; Hail, Mark E.; Wu, Yifu; Shaw, Sunil K.

doi:10.1006/abio.1999.4095

Cited by 22 publications

(24 citation statements)

References 15 publications

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“…Molar extinction values used for FXI subunit and PK were 83,780 and 92,030, respectively. Partial specific volumes (v ) were calculated from the sequences using "Sednterp" (20) and adjusted (21) for glycosylation levels of 5% for FXI and FXI mutants and 15.5% for PK (12). The pK d value is defined as the negative base 10 logarithm of the monomer-dimer dissociation constant in molar units.…”

Section: Methodsmentioning

confidence: 99%

Factor XI Homodimer Structure Is Essential for Normal Proteolytic Activation by Factor XIIa, Thrombin, and Factor XIa

Sinha

Shikov

et al. 2008

Journal of Biological Chemistry

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Coagulation factor XI (FXI) is a covalent homodimer consisting of two identical subunits of 80 kDa linked by a disulfide bond formed by Cys-321 within the Apple 4 domain of each subunit. Because FXI C321S is a noncovalent dimer, residues within the interface between the two subunits must mediate its homodimeric structure. The crystal structure of FXI demonstrates formation of salt bridges between Lys-331 of one subunit and Glu-287 of the other subunit and hydrophobic interactions at the interface of the Apple 4 domains involving Ile-290, Leu-284, and Tyr-329. FXI C321S , FXI C321S,K331A , FXI C321S,E287A , FXI C321S,I290A , FXI C321S,Y329A , FXI C321S,L284A , FXI C321S,K331R , and FXI C321S,H343A were expressed in HEK293 cells and characterized using size exclusion chromatography, analytical ultracentrifugation, electron microscopy, and functional assays. Whereas FXI C321S and FXI C321S,H343A existed in monomer/ dimer equilibrium (K d ϳ 40 nM), all other mutants were predominantly monomers with impaired dimer formation by analytical ultracentrifugation (K d ‫؍‬ 3-38 M). When converted to the active enzyme, FXIa, all the monomeric mutants activated FIX similarly to wild-type dimeric FXIa. In contrast, these monomeric mutants could not be activated efficiently by FXIIa, thrombin, or autoactivation in the presence of dextran sulfate. We conclude that salt bridges formed between Lys-331 of one subunit and Glu-287 of the other together with hydrophobic interactions at the interface, involving residues Ile-290, Leu-284, and Tyr-329, are essential for homodimer formation. The dimeric structure of FXI is essential for normal proteolytic activation of FXI by FXIIa, thrombin, or FXIa either in solution or on an anionic surface but not for FIX activation by FXIa in solution.Factor XI (FXI), 3 the zymogen form of a serine protease coagulation enzyme that is essential for normal hemostasis, is activated either by FXIIa or by thrombin or by autoactivation (1, 2). Once converted to FXIa, the enzyme recognizes its natural macromolecular substrate, FIX, the Ca 2ϩ -dependent activation of which requires the exposure of a substrate-binding site within the Apple 2 (A2) and/or Apple 3 (A3) domains of FXIa and the ␥-carboxyglutamic acid domain of FIX, as well as an extended, macromolecular substrate-binding exosite in the protease domain of FXIa (3-9). The activation of FIX to FIXa␤ involves two cleavages by FXIa, one after Arg-145 and the other after Arg-180, thereby releasing an 11-kDa activation peptide (3,4,10). FIX is also activated to FIXa␤ by the tissue factorFVIIa complex (11).FXI and plasma prekallikrein (PK) are 58% identical in their amino acid sequences, and the domain structures of the two molecules are very similar, with each molecule containing four homologous apple (A1-A4) domains (12). The high homology between the heavy chain of FXI and PK indicates a common origin of these two zymogens, in contrast to FXII and other coagulation factors (13,14). However, FXI is a homodimer of two identical subunits joined by a disu...

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Section: Methodsmentioning

confidence: 99%

Factor XI Homodimer Structure Is Essential for Normal Proteolytic Activation by Factor XIIa, Thrombin, and Factor XIa

Sinha

Shikov

et al. 2008

Journal of Biological Chemistry

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“…This analysis is dependent on comparing the RI and UV areas of baseline-resolved peaks to calibration curves for each component. Absorptive losses of any of the components will influence the final 1 To whom reprint requests should be addressed at current address: Amgen, Inc., 4000 Nelson Road, MS AC-3A, Longmont, CO 80503. Fax: (303) 938-6241.…”

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confidence: 99%

“…Empirical methods such as size-exclusion chromatography or dynamic light scattering can be run under physiological conditions, but only measure hydrodynamic radius. Interpreting hydrodynamic radius in terms of molecular mass for conjugated proteins has been elusive (1,2). Sedimentation equilibrium (SE) ultracentrifugation is another useful technique; however, it relies on analysis of extremely pure samples that are stable for the length of the analysis (i.e., a few days if multiple speeds are used) (1,3).…”

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confidence: 99%

Online Size-Exclusion High-Performance Liquid Chromatography Light Scattering and Differential Refractometry Methods to Determine Degree of Polymer Conjugation to Proteins and Protein–Protein or Protein–Ligand Association States

Kendrick

Kerwin

Chang

et al. 2001

Analytical Biochemistry

124

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“…However, in the present study, considering bioinformatical model and predicted glycosylation sites along with biophysical analysis revealed that the glycosylated vOX2 should have a molecular mass of 32.0-33.6 kDa, corresponding to 30-36% glycosylation. This number is comparable to other glycosylated proteins participating in the immunological response such as CD80 family (Fairman et al, 1999). In our suggested model, high ERRAT score (96.3%) besides fine matching of bioinformatical and biophysical data, could provide more confidence on accuracy of bioinformatically determined three-dimensional model and infers that PD-L1 selection as a template is satisfactory.…”

Section: Discussionmentioning

confidence: 58%

Structural properties of a viral orthologue of cellular CD200 protein: KSHV vOX2

et al. 2015

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Kaposi׳s sarcoma-associated herpesvirus (KSHV) vOX2 is a cell surface glycoprotein expressed during viral lytic replication to suppress host inflammatory reactions. Here we have characterised vOX2 with biochemical, biophysical and bioinformatics tools and as a result propose a 3-dimensional model for vOX2 based on structural and functional homology with the PD-L1 protein. To validate this model, vOX2 was characterised by analytical ultracentrifugation (AUC) and circular dichroism spectroscopy (CD). The results identified the potential glycosylation sites and revealed that vOX2 is predominantly a beta-folded molecule with an RGD adhesion motif exposed on the C-terminal domain. The protein exists in monomer-dimer equilibrium similar to its IgV-type folded homologues, with 30-36% glycosylation and the molecular weight of the extracellular fragment of molecule is 32.0-33.6 kDa, much less than 50 kDa. Thus, the structural similarity to PD-L1 verifies its immunomodulatory potential and the RGD motif suggests an adhesive capacity.

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Molecular Weights of CTLA-4 and CD80 by Sedimentation Equilibrium Ultracentrifugation

Cited by 22 publications

References 15 publications

Factor XI Homodimer Structure Is Essential for Normal Proteolytic Activation by Factor XIIa, Thrombin, and Factor XIa

Factor XI Homodimer Structure Is Essential for Normal Proteolytic Activation by Factor XIIa, Thrombin, and Factor XIa

Online Size-Exclusion High-Performance Liquid Chromatography Light Scattering and Differential Refractometry Methods to Determine Degree of Polymer Conjugation to Proteins and Protein–Protein or Protein–Ligand Association States

Structural properties of a viral orthologue of cellular CD200 protein: KSHV vOX2

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