The role of mannosylated enzyme and the mannose receptor in enzyme replacement therapy.

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.

Effect of genetic background on glycosylation heterogeneity in human antithrombin produced in the mammary gland of transgenic goats.

Glycosylation is involved in the correct folding, targeting, bioactivity and clearance of therapeutic glycoproteins. With the development of transgenic animals as expression systems it is important to understand the impact of different genetic backgrounds and lactations on glycosylation. We have evaluated the glycosylation of recombinant antithrombin produced in several transgenic goat lines, from cloned animals and from different types of lactation including induced lactations. Our results show glycosylation patterns from the protein expressed in animals, derived from the same founder goat, are mostly comparable. Furthermore, the protein expressed in two cloned goats had highly consistent oligosaccharide profiles and similar carbohydrate composition. However, there were significantly different oligosaccharide profiles from the proteins derived from different founder goats. Artificial induction of lactation did not have significant effects on overall carbohydrate structures when compared to natural lactation. The only major difference was that recombinant antithrombin from induced lactations contained a slightly higher ratio of N-acetylneuraminic acid to N-glycolylneuraminic acid and less amount of oligosaccharides containing N-glycolylneuraminic acid. The oligosaccharides from all animals were a mixture of high mannose-, hybrid- and complex-type oligosaccharides. Sialic acid was present as alpha-2,6-linkage and no alpha-1,3-linked galactose was observed. These results indicate that transgenic animals with closely related genetic backgrounds express recombinant protein with comparable glycosylation.

The effect of posttranslational modifications on the in vitro activity of recombinant human thyroid-stimulating hormone.

Posttranslational modification can influence the biologic activity of recombinant proteins. The effects of beta-subunit C-terminal truncation, oligosaccharide heterogeneity, and chemical oxidation on the in vitro activity of recombinant human thyroid-stimulating hormone (rhTSH) were investigated. beta-Subunit C-terminal truncation up to residue 113 did not effect the in vitro activity of the hormone. The relationship between the heterogeneity of oligosaccharide structures on rhTSH and specific activity of the glycoprotein hormone was also examined. Oligosaccharide profiles were generated for preparations of rhTSH containing similar sialic acid levels. A weak correlation was observed between relative levels of monosialylated biantennary, bisialylated biantennary, and trisialylated triantennary oligosaccharide species and in vitro activity of the recombinant hormone (p < 0.05). To examine the effect of chemically induced methionine oxidation on the activity of rhTSH, the hormone was treated with tert-butyl hydroperoxide and then characterized. Using peptide mapping and mass spectrometry, the degree of oxidation of the five methionine residues within rhTSH was measured. Met-71 in the alpha-subunit was the most susceptible to oxidation whereas Met-9 in the beta-subunit was the most resistant. Also, after tert-butyl hydroperoxide treatment, levels of oxidation of Met-32 in the beta-subunit, and Met-29 and Met-47 in the alpha-subunit were less than half of that observed for Met-71. The in vitro activity of rhTSH initially declined with increasing oxidation; however, the loss in activity plateaued at approximately 50% of the control sample activity. In summary, despite the possible effects that posttranslational modifications may have on the bioactivity of a protein, a limited degree of variation in bioactivity was observed for the rhTSH preparations described in this study.

Comparison of two in vitro methods for the measurement of recombinant human TSH bioactivity.

Thyroid stimulating hormone (TSH), a pituitary glycoprotein hormone, is a potent inducer of intracellular cAMP production. Two methods for measuring TSH bioactivity were evaluated and compared. One assay is based on using a radioimmunoassay (RIA) to measure the recombinant human TSH-induced increase in cAMP using a bovine thyroid membrane isolate. The other is based on a Chinese hamster ovary (CHO) cell line that has been transfected with the TSH receptor and a cAMP-responsive luciferase reporter. The within-assay coefficient of variation for the membrane-based assay was determined to be approximately 35% compared with approximately 25% for the cell-based assay. Twenty-one preparations of recombinant human TSH (rhTSH) were tested using both methods. No significant difference was detected between the data sets and no assay bias was present. Both assay systems provide a suitable means for measuring the activity of rhTSH. The advantage of the membrane-based assay is the relatively small quantity of TSH needed for analysis. However, the average time required to analyse a sample using the membrane-based method was more than twice as long as that needed to test a sample in the cell-based assay. Other advantages of the cell-based method include the use of a 96-well format, which facilitates the analysis of several concentrations of rhTSH within one assay plate, and the use of a non-radioactive endpoint.