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MMCC Results Volume 15 No. 2 1 June 2006

Protein Design and Engineering

A designed glycoprotein analogue of Gc-MAF exhibits native-like phagocytic activity.

Federica Bogani, Elizabeth McConnell, Lokesh Joshi, Yung Chang, and Giovanna Ghirlanda* [Arizona State Univ.]

J. Am. Chem. Soc.

128, 7142 -7143 (2006)

De novo protein engineering is used to develop a mini-protein analogue of Gc-MAF, a glycoprotein involved in the immune system activation that showed anticancer activity in mice. Molecular modeling tools are used in conjunction with structural analysis to splice the glycosylated loop onto a stable three-helix bundle. The resulting 69-residue model peptide, MM1, is synthesized by solid-phase synthesis both in the aglycosylated and the glycosylated (GalNAc-MM1) form. GalNAc-MM1 provides a framework for the development of mutants with increased activity that is used in place of Gc-MAF as an immunomodulatory agent in therapy.

 

Source: http://www.ccl.net/cca/documents/MMCC_Results/MMCC_V15_i2_2006.pdf

MMCC Results Volume 15 No. 2 1 June 2006

 

 

Modulation of protein stability by O-glycosylation in a designed Gc-MAF analog

The post-translational modification of proteins by the covalent attachment of carbohydrates to specific side chains, or glycosylation, is emerging as a crucial process in modulating the function of proteins. In particular, the dynamic processing of the oligosaccharide can correlate with a change in function. For example, a potent macrophage-activating factor, Gc-MAF, is obtained from serum vitamin D binding protein (VDBP) by stepwise processing of the oligosaccharide attached to Thr 420 to the core ?-GalNAc moiety. In previous work we designed a miniprotein analog of Gc-MAF, MM1, by grafting the glycosylated loop of Gc-MAF on a stable scaffold. GalNAc-MM1 showed native-like activity on macrophages (Bogani 2006, J. Am. Chem. Soc. 128 7142–43).

Here, we present data on the thermodynamic stability and conformational dynamics of the mono- and diglycosylated forms. We observed an unusual trend: each glycosylation event destabilized the protein by about 1 kcal/mol. This effect is matched by an increase in the mobility of the glycosylated forms, as evaluated by molecular dynamics simulations. An analysis of the solvent-accessible surface area shows that glycosylation causes the three-helix bundle to adopt conformations in which the hydrophobic residues are more solvent exposed. The number of hydrophobic contacts is also affected.

These two factors, which are ultimately explained with a change in occupancy for conformers of specific side chains, may contribute to the observed destabilization.

 

Gc Protein (Vitamin D-binding Protein): Gc Genotyping and GcMAF Precursor Activity (Abstract)

The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5? for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity.

 

Preparation of GcMAF and its structural characterization and biological activities (Abstract)

Background: Gc protein has been reported to be a precursor of Gc protein-derived macrophage activation factor (GcMAF) in the inflammation- primed macrophage activation cascade. An inducible beta-galactosidase of B cells and neuraminidase of T cells convert Gc protein to GcMAF.

Materials and Methods: Gc protein from human serum was purred using 25(OH)D3affinity column chromatography and modified to GcMAF using immobilized glycosidases (beta-galactosidase and neuraminidase) The sugar moiety structure of GcMAF was characterized by lectin blotting by Helix pomatia agglutinin. The biological activities of GcMAF were evaluated by a superoxide generation assay and a phagocytosis assay. Results: We successfully purified Gc protein from human serum. GcMAF was detected by lectin blotting and showed a high biological activity.

Conclusion: Our results support the importance of the terminal N-acetylgalactosamine moiety in the GeMAF-mediated macrophage activation cascade, and the existence of constitutive GcMAF in human serum. These preliminary data are important for designing small molecular GcMAF mimics.

 

Cloned GcMAF in cancer, HIV-infection and osteopetrosis

United States Patent 6410269 - details will follow shortly
Vitamin D-binding protein (Gc protein) and its small domain (approximately ? of the Gc peptide also known as domain III) were cloned via a baculovirus vector. The cloned Gc protein and the cloned domain (Cd) peptide were treated with immobilized ?-galactosidase and sialidase to yield macrophage activating factors, GcMAFc and CdMAF, respectively. These cloned macrophage activating factors and GcMAF are to be used for therapy of cancer, HIV-infection and osteopetrosis, and may also be used as adjuvants for immunization and vaccination.

Source: http://www.freepatentsonline.com/6410269.html