Engineering of the Saccharomyces cerevisiae yeast strain with multiple chromosome-integrated genes of human alpha-fetoprotein and its high-yield secretory production, purification, structural and functional characterization.

Alpha-fetoprotein (AFP) is a biological drug candidate of high medicinal potential in the treatment of autoimmune diseases, cancer, and regenerative medicine. Large-scale production of recombinant human alpha-fetoprotein (rhAFP) is desirable for structural and functional studies and applied research. In this study we cloned and expressed in the secreted form wild-type glycosylated human rhAFP and non-glycosylated mutant rhAFP(0) (N233S) in the yeast strain Saccharomyces cerevisiae with multiple chromosome-integrated synthetic human AFP genes. RhAFP and rhAFP(0) were successfully produced and purified from the culture liquids active naturally folded proteins. Elimination of the glycosylation by mutation reduced rhAFP(0) secretion about threefold as compared to the wild-type protein showing critical role of the N-linked glycan for heterologous protein folding and secretion. Structural similarity of rhAFP and rhAFP(0) with natural embryonic eAFP was confirmed by circular dichroism technique. Functional tests demonstrated similar type of tumor suppressive and immunosuppressive activity for both recombinant species rhAFP and rhAFP(0) as compared to natural eAFP. It was documented that both types of biological activities attributed to rhAFP and rhAFP(0) are due to the fast induction of apoptosis in tumor cells and mitogen-activated lymphocytes. Despite the fact that rhAFP and rhAFP(0) demonstrated slightly less effective tumor suppressive activity as compared to eAFP but rhAFP(0) had produced statistically notable increase in its ability to induce inhibition of in vitro lymphocyte proliferation as compared to the glycosylated rhAFP and eAFP. We conclude that N-linked glycosylation of rhAFP is required for efficient folding and secretion. However the presence of N-linked sugar moiety was shown to be unimportant for tumor suppressive activity but was critically important for its immunoregulative activity which demonstrates that different molecular mechanisms are involved in these two types of biological functional activities attributed to AFP.

A mouse model for Sorsby fundus dystrophy.

PURPOSE: Sorsby fundus dystrophy (SFD) is a rare, late-onset macular dystrophy caused by mutations in the tissue inhibitor of metalloproteinases-3 (TIMP3) gene. The known mutations introduce potentially unpaired cysteine residues in the C terminus of the protein and result in the formation of higher-molecular-weight protein complexes of as yet unknown composition and functional consequences in the pathologic course of SFD. To facilitate in vivo investigation of mutant TIMP3, the authors generated a knock-in mouse carrying a disease-related Ser156Cys mutation in the orthologous murine Timp3 gene. METHODS: Site-directed mutagenesis and homologous recombination in embryonic stem (ES) cells was used to generate mutant ES cells carrying the Timp3(S156C) allele. Chimeric animals were obtained, of which two displayed germline transmission of the mutated allele. Molecular genetic, biochemical, electron microscopic, and electrodiagnostic techniques were used for characterization. RESULTS: At 8 months of age, knock-in mice showed abnormalities in the inner aspect of Bruch's membrane and in the organization of the adjacent basal microvilli of the retinal pigment epithelium (RPE). Changes resembling those in the mutant animals were also present to some extent in normal littermates, but only at an advanced age of 30 months. Long-term electrodiagnostic recordings indicated normal retinal function throughout life. The biochemical characteristics of the mutant protein appear similar in humans and knock-in mice, suggesting common molecular pathways in the two species. The localization of the mutant protein in the eye is normal, although there is evidence of increased Timp3 levels in Bruch's membrane of mutant animals. CONCLUSIONS: The knock-in mice display early features of age-related changes in Bruch's membrane and the RPE that may represent the primary clinical manifestations of SFD. In addition, our immunolabeling studies and biochemical data support a model proposing that site-specific excess rather than absence or deficiency of functional Timp3 may be the primary consequence of the known Timp3 mutations.

Sorsby fundus dystrophy mutation Timp3(S156C) affects the morphological and biochemical phenotype but not metalloproteinase homeostasis.

The tissue inhibitor of metalloproteinases-3 (TIMP3) is a multifunctional protein tightly associated with the extracellular matrix (ECM). A specific type of mutation in TIMP3 which results in potentially unpaired cysteine residues at the C-terminus of the protein has been shown to cause Sorsby fundus dystrophy (SFD), an autosomal dominant retinopathy of late onset. An early finding in SFD is a striking accumulation of protein and lipid material in Bruch's membrane, a multilayered ECM structure located between the choroid and the RPE. To study the molecular mechanisms underlying SFD pathology, we recently generated two mouse lines, one deficient in Timp3 (Timp3(-/-)) and one carrying an SFD-related mutation in the orthologous murine Timp3 gene (Timp3(S156C/S156C)). We now established immortalized fibroblast cells from the mutant mouse strains and provide evidence that the various cell lines display distinct morphological and physiological features that are dependent on the mutational status of the Timp3 protein in the secreted ECM. We show that matrix metalloproteinase (MMP) activity and inhibitory properties of Timp3 are not affected by the SFD-associated mutation. We further demonstrate that Timp3(S156C) protein accumulates in the ECM of the mutant fibroblast cells and that this accumulation is not due to a prolonged turnover rate of mutant vs. normal Timp3. We also show that the relative abundance of mutant and normal Timp3 in the ECM has no measurable effects on cellular phenotypes. Together, these findings suggest (i) a functional role of normal Timp3 in pathways determining cellular morphology and (ii) a loss of this particular function as a consequence of the Ser156Cys mutation. We therefore hypothesize that SFD pathogenesis is due to a loss-of-function mutation in TIMP3.