Characterization and expression of proprotein convertases in CHO cells: Efficient proteolytic maturation of human bone morphogenetic protein-7.

Bone morphogenetic protein-7 (BMP-7) is synthesized as a precursor that requires proteolytic cleavage of the propeptide by proprotein convertases (PCs) for its functional activity. A high-level expression of BMP-7 in CHO cells (CHO-BMP-7) resulted in secretion of a mixture of inactive precursor and active BMP-7. In an effort to achieve efficient processing of BMP-7 in CHO cells, PCs responsible for cleavage of the precursors in CHO cells were characterized. Analysis of the mRNA expression levels of four PCs (furin, PACE4, PC5/6, and PC7) revealed that only furin and PC7 genes are expressed in CHO-BMP-7 cells. Specific inhibition of the PCs by hexa-D-arginine (D6R) or decanoyl-RVKR-chloromethyl ketone (RVKR-CMK) further revealed that furin is mainly responsible for the proteolytic processing of BMP-7. To identify a more efficient PC for BMP-7 processing, the four PC genes were transiently expressed in CHO-BMP-7 cells, respectively. Among these, PC5/6 was found to be the most efficient in BMP-7 processing. Stable overexpression of PC5/6ΔC, a secreted form of PC5/6, significantly improved mature BMP-7 production in CHO-BMP-7 cells. When the maximum BMP-7 concentration was obtained in the culture of CHO-BMP-7 cells, approximately 88% of BMP-7 was unprocessed. In contrast, no precursor was found in the culture of PC5/6ΔC-overexpressing cells (clone #97). Furthermore, the in vitro biological activity of the mature BMP-7 from PC5/6ΔC-overexpressing cells was comparable to that from CHO-BMP-7 cells. Taken together, the present results indicate that overexpression of PC5/6ΔC in CHO-BMP-7 cells is an efficient means of increasing the yield of BMP-7.

Functional analysis of mutations in UDP-galactose-4-epimerase (GALE) associated with galactosemia in Korean patients using mammalian GALE-null cells.

Galactosemia is caused by defects in the galactose metabolic pathway, which consists of three enzymes, including UDP-galactose-4-epimerase (GALE). We previously reported nine mutations in Korean patients with epimerase-deficiency galactosemia. In order to determine the functional consequences of these mutations, we expressed wild-type and mutant GALE proteins in 293T cells. GALE(E165K) and GALE(W336X) proteins were unstable, had reduced half-life, formed aggregates and were partly degraded by the proteasome complex. When expressed in GALE-null ldlD cells GALE(E165K), GALE(R239W), GALE(G302D) and GALE(W336X) had no detectable enzyme activity, although substantial amounts of protein were detected in western blots. The relative activities of other mutants were lower than that of wild-type. In addition, unlike wild-type, GALE(R239W) and GALE(G302D) were not able to rescue galactose-sensitive cell proliferation when stably expressed in ldlD cells. The four inactive mutant proteins did not show defects in dimerization or affect the activity of other mutant alleles identified in patients. Our observations show that altered protein stability is due to misfolding and that loss or reduction of enzyme activity is responsible for the molecular defects underlying GALE-deficiency galactosemia.

Molecular and biochemical characterization of the GALK1 gene in Korean patients with galactokinase deficiency.

Galactokinase (GALK) deficiency is an autosomal recessive disorder characterized by elevation of blood galactose concentration and diminished galactose-1-phosphate, leading to the production of galactitol. To investigate the molecular defects of GALK1 gene and the biochemical characteristics of their mutant proteins, PCR-direct sequencing and in vitro expression analysis in Cos7 cells were performed in five Korean patients with GALK deficiency galactosemia. Four missense mutations (p.G137R, p.R256W, p.R277Q, and p.V281M) and one small insertion (c.850_851insG) were identified. Among four patients with severely reduced GALK activity, two were found to be homozygotes for p.R256W and the other two were compound heterozygotes for different molecular defects (p.G137R/p.R277Q and p.V281M/c.850_851insG). One Patient with moderately decreased GALK activity was heterozygous for p.R256W. Expression analysis in Cos7 cells confirmed that each of the mutations resulted in reduction of GALK activity and caused GALK deficiency.