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Published on: January 8, 2014
Structural characterization of a recombinant CD4-IgG hybrid molecule
R J Harris1, K L Wagner, M W Spellman
1Department of Medicinal and Analytical Chemistry, Genentech, Inc., South San Francisco, CA 94080.
Insights
This study characterizes CD4-IgG, an immunoadhesin combining CD4 binding with IgG properties. Structural analysis confirmed its expected features, including glycosylation and disulfide bonds.
Area of Science:
- Biochemistry
- Immunology
- Molecular Biology
Background:
- CD4 is a crucial co-receptor in T-cell activation.
- Immunoadhesins offer potential therapeutic advantages by combining binding specificity with effector functions.
Purpose of the Study:
- To structurally characterize CD4-IgG, an immunoadhesin.
- To confirm the fusion of human CD4 domains with human IgG-1 Fc domains.
- To validate the expected molecular features of this novel construct.
Main Methods:
- Peptide mapping using tryptic digestion.
- Analysis of tryptic peptides via chromatography.
- Deglycosylation and mass spectrometry to confirm glycosylation sites.
Main Results:
- Structural analysis confirmed 98.8% of the expected CD4-IgG structure.
- Presence of Asn-linked oligosaccharides at Asn257 was confirmed.
- Four intrachain and two interchain disulfide bonds were identified, consistent with IgG structure.
Conclusions:
- CD4-IgG possesses structural integrity mirroring both soluble CD4 and IgG Fc domains.
- The molecule's structure is well-defined, supporting its potential for therapeutic applications.
- The characterization provides a foundation for further functional studies of CD4-IgG.
Abstract:
CD4-IgG is a homodimer of a hybrid polypeptide consisting of the two amino-terminal domains (residues 1-180) of human CD4 fused to the hinge region and the second and third constant-sequence (CH2 and CH3) Fc domains (residues 216-441) of human immunoglobulin G (IgG-1). This antibody-like molecule, termed an immunoadhesin, was produced in an effort to combine the binding specificity of CD4 with several potentially desirable properties of IgG molecules [Capon et al. (1989) Nature 337, 525-531]. The structural characteristics of the molecule have been evaluated to demonstrate that CD4-IgG has the same features as the N-terminal region of soluble CD4, while retaining those expected for the Fc portion of human IgG. Identification of peptides recovered from the tryptic map confirmed 98.8% of the expected structure of CD4-IgG. The detection of glucosamine in peptides containing Asn257 and the retention time shift of this tryptic peptide after deglycosylation confirmed the presence of Asn-linked oligosaccharides at this position. Four pairs of intrachain and two interchain disulfide bonds were also established.
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