Recombinant human antithrombin (rhAT) produced in transgenic goat milk was purified to greater than 99%. The specific activity of the rhAT was identical to human plasma–derived AT (phAT) in an in vitro thrombin inhibition assay. However, rhAT had a fourfold higher affinity for heparin than phAT. The rhAT was analyzed and compared with phAT by reverse phase high-performance liquid chromatography, circular dichroism, fluorophore-assisted carbohydrate electrophoresis (FACE), amino acid sequence, and liquid chromatography/mass spectrography peptide mapping. Based on these analyses, rhAT was determined to be structurally identical to phAT except for differences in glycosylation. Oligomannose structures were found on the Asn 155 site of the transgenic protein, whereas only complex structures were observed on the plasma protein. RhAT contained a GalNAc for galactose substitution on some N-linked oligosaccharides, as well as a high degree of fucosylation. RhAT was less sialylated than phAT and contained both N-acetylneuraminic and N-glycolylneuraminic acid. We postulate that the increase in affinity for heparin found with rhAT resulted from the presence of oligomannose-type structures on the Asn 155 glycosylation site and differences in sialylation.
Lysosomal acid lipase (LAL) is the critical enzyme for the hydrolysis of triglycerides (TGs) and cholesteryl esters (CEs) in lysosomes. LAL defects cause Wolman disease (WD) and CE storage disease (CESD). An LAL null (lal-/-) mouse model closely mimics human WD/CESD, with hepatocellular, Kupffer cell and other macrophage, and adrenal cortical storage of CEs and TGs. The effect on the cellular targeting of high-mannose and complex oligosaccharide-type oligosaccharide chains was tested with human LAL expressed in Pichia pastoris (phLAL) and CHO cells (chLAL), respectively. Only chLAL was internalized by cultured fibroblasts, whereas both chLAL and phLAL were taken up by macrophage mannose receptor (MMR)-positive J774E cells. After intraperitoneal injection into lal-/- mice, phLAL and chLAL distributed to macrophages and macrophage-derived cells of various organs. chLAL was also detected in hepatocytes. Ten injections of either enzyme over 30 d into 2- and 2.5-mo-old lal-/- mice produced normalization of hepatic color, decreased liver weight (50%-58%), and diminished hepatic cholesterol and TG storage. Lipid accumulations in macrophages were diminished with either enzyme. Only chLAL cleared lipids in hepatocytes. Mice double homozygous for the LAL and MMR deficiences (lal-/-;MMR-/-) showed phLAL uptake into Kupffer cells and hepatocytes, reversal of macrophage histopathology and lipid storage in all tissues, and clearance of hepatocytes. These results implicate MMR-independent and mannose 6-phosphate receptor-independent pathways in phLAL uptake and delivery to lysosomes in vivo. In addition, these studies show specific cellular targeting and physiologic effects of differentially oligosaccharide-modified human LALs mediated by MMR and that lysosomal targeting of mannose-terminated glycoproteins occurs and storage can be eliminated effectively without MMR.
The presence and specific structures of the oligosaccharides on TSH have been shown to be important for its production and bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the biological activity and metabolic clearance of a preparation of purified recombinant human (rh) TSH derived from a stable transfectant of Chinese hamster ovary cells. Carbohydrate compositional analysis of this rTSH showed it to be more highly sialylated than a nonrecombinant, cadaver-derived pituitary hTSH. In addition, no N-acetyl galactosamine was detectable in rhTSH, which implies the absence of terminal sulfate moieties, both of which are present in pituitary-derived TSH. The immunologic activity and porcine TSH receptor-binding activity of the preparation of rhTSH were 3- to 4-fold lower than those of a standard pituitary hTSH. The rhTSH showed a maximum stimulatory activity similar to that of pituitary hTSH in two different in vitro bioassays. However, rhTSH elicited about 3-fold and 5-fold less cAMP than pituitary TSH after stimulation of adenylyl cyclase in bovine thyroid membranes and the rat FRTL-5 cell line, respectively. Removal of sialic acid did not alter the immunologic activity of rhTSH. However, the potencies of rhTSH in receptor-binding, adenylyl cyclase, and FRTL-5 assays were increased 2.4-, 2.6- and 26.7-fold, respectively after sialic acid removal. These data suggest that the in vitro biological activity of rhTSH is influenced by its highly sialylated oligosaccharide chains. The rhTSH had a 2-fold lower metabolic clearance rate than pituitary TSH, resulting in a greater than 10-fold higher serum concentration of rhTSH at 3 h as compared to pituitary hTSH. After sialic acid removal, the rhTSH was cleared faster (7.5-fold) than pituitary hTSH, showing that its longer plasma half-life was due to its higher sialylation. Biologically active rhTSH should be of clinical value in the diagnosis and treatment of patients with thyroid cancer and as a pure hTSH reference preparation.
We have genetically engineered a cell line, and developed a reproducible process, for the expression and purification of biologically active recombinant human thyroid stimulating hormone (rhTSH).rhTSH was expressed by co-transfecting a human alpha-subunit cDNA with a human beta-subunit partial genomic clone into Chinese Hamster Ovary (CHO) cells. Stable transfectants which expressed high levels of rhTSH were selected, and subsequently cultured on microcarrier beads. The rhTSH-containing media, produced under serum-free conditions, was clarified and purified by a combination of ion exchange, dye and gel filtration chromatographies. Individual step recoveries were greater than 90% with the exception of a very conservative pooling of the final gel filtration step (78% recovery) that resulted in a cumulative yield of 54% for the purification process. Purity of the final bulk material was judged to be > 99% by SDS polyacrylamide gel electrophoresis (SDS-PAGE), reverse phase HPLC, and size exclusion chromatography. Initial characterization of the oligosaccharide composition indicated the presence of partially sialylated bi- and triantenary complex oligosaccharides. Purified rhTSH was active in a thyroid membrane bioactivity assay with a specific activity of 8.2 IU/mg. The in vivo activity of rhTSH in cynomolgus monkeys appeared to be equal to or greater than that reported for bovine TSH (bTSH) in human subjects. The rapid clearance phase half-life of rhTSH was approximately 35 minutes while the post-distribution phase half life was approximately 9.8 hours. Furthermore, the monkeys showed cumulative increases in minimum plasma rhTSH levels when given three daily intramuscular (IM) rhTSH injections; a phenomenon not observed when bTSH had been administered to humans. The rhTSH showed no evidence of toxic or adverse effects when administered at doses up to 7.2 IU/kg and 0.52 IU/kg in rat and monkey, respectively. These are 50X and 4X multiples of the bTSH doses of 0.143 IU/kg (10 IU/70kg) previously administered to humans.
Recombinant human antithrombin (rhAT) produced in transgenic goat milk was purified to greater than 99%. The specific activity of the rhAT was identical to human plasma–derived AT (phAT) in an in vitro thrombin inhibition assay. However, rhAT had a fourfold higher affinity for heparin than phAT. The rhAT was analyzed and compared with phAT by reverse phase high-performance liquid chromatography, circular dichroism, fluorophore-assisted carbohydrate electrophoresis (FACE), amino acid sequence, and liquid chromatography/mass spectrography peptide mapping. Based on these analyses, rhAT was determined to be structurally identical to phAT except for differences in glycosylation. Oligomannose structures were found on the Asn 155 site of the transgenic protein, whereas only complex structures were observed on the plasma protein. RhAT contained a GalNAc for galactose substitution on some N-linked oligosaccharides, as well as a high degree of fucosylation. RhAT was less sialylated than phAT and contained both N-acetylneuraminic and N-glycolylneuraminic acid. We postulate that the increase in affinity for heparin found with rhAT resulted from the presence of oligomannose-type structures on the Asn 155 glycosylation site and differences in sialylation.
Recombinant human PRL was produced in a murine C127 cell expression system and purified to greater than 97% homogeneity using anion and cation exchange chromatography. This material was biologically equivalent to pituitary-derived PRL in both an enzyme-linked immunosorbent assay and the Nb2 lymphoma cell proliferation assay. The predominant PRL forms were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting as being 23 and 25 kilodaltons (kDa). These mass values were confirmed by electrospray mass spectroscopy. Glycosidase digestions indicated that the 25-kDa PRL is N-glycosylated and sialylated, whereas 23-kDa PRL is nonglycosylated. Glycosylated and nonglycosylated forms of the hormone were individually purified to greater than 95% homogeneity using novel cation exchange chromatography. Isoelectric focusing demonstrated that both forms consist of multiple charge isomers, with the charge heterogeneity of the glycosylated form primarily due to differences in sialylation. Monosaccharide analysis of the glycosylated form suggested a minimal complex oligosaccharide chain that may be fucosylated and partially sialylated. Oligosaccharide mol wt were determined by electrospray ionization mass spectroscopy. Analysis of the oligosaccharides by fluorophore-assisted carbohydrate electrophoresis indicated that bi- and triantennary oligosaccharide forms are predominant and have multiple combinations of terminal sialylation. Both forms of PRL were active in the Nb2 lymphoma cell proliferation assay; however, the 23-kDa nonglycosylated form was 3-4 times more active in this assay than the 25-kDa glycosylated form.
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