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Oligosaccharide structures and the degree of sialylation can cause variability among detection cells when using high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) technology for oligosaccharide characterization. That variability can [...]

To enhance the delivery of rhGAA (recombinant GAA, where GAA stands for acid α-glucosidase) to the affected muscles in Pompe disease, the carbohydrate moieties on the enzyme were remodelled to exhibit a high affinity ligand for the CI-MPR (cation-independent M6P receptor, where M6P stands for mannose 6-phosphate). This was achieved by chemically conjugating on to rhGAA, a synthetic oligosaccharide ligand bearing M6P residues in the optimal configuration for binding the receptor. The carbonyl chemistry used resulted in the conjugation of approx. six synthetic ligands on to each enzyme. The resulting modified enzyme [neo-rhGAA (modified recombinant human GAA harbouring synthetic oligosaccharide ligands)] displayed near-normal specific activity and significantly increased affinity for the CI-MPR. However, binding to the mannose receptor was unaffected despite the introduction of additional mannose residues in neo-rhGAA. Uptake studies using L6 myoblasts showed neo-rhGAA was internalized approx. 20-fold more efficiently than the unmodified enzyme. Administration of neo-rhGAA into Pompe mice also resulted in greater clearance of glycogen from all the affected muscles when compared with the unmodified rhGAA. Comparable reductions in tissue glycogen levels in the Pompe mice were realized using an approx. 8-fold lower dose of neo-rhGAA in the heart and diaphragm and an approx. 4-fold lower dose in the skeletal muscles. Treatment of older Pompe mice, which are more refractory to enzyme therapy, with 40 mg/kg neo-rhGAA resulted in near-complete clearance of glycogen from all the affected muscles as opposed to only partial correction with the unmodified rhGAA. These results demonstrate that remodelling the carbohydrate of rhGAA to improve its affinity for the CI-MPR represents a feasible approach to enhance the efficacy of enzyme replacement therapy for Pompe disease.

Posttranslational modification can influence the biologic activity of recombinant proteins. The effects of β-subunit C-terminal truncation, oligosaccharide heterogeneity, and chemical oxidation on the in vitroactivity of recombinant human thyroid-stimulating hormone (rhTSH) were investigated. β-Subunit C-terminal truncation up to residue 113 did not effect the in vitroactivity 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 vitroactivity 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 α-subunit was the most susceptible to oxidation whereas Met-9 in the β-subunit was the most resistant. Also, after tert-butyl hydroperoxide treatment, levels of oxidation of Met-32 in the β-subunit, and Met-29 and Met-47 in the α-subunit were less than half of that observed for Met-71. The in vitroactivity 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.

Two homologous proteins, murine (m) and human (h) granulocyte–macrophage colony–stimulating–factor (GM–CSF), were differentially glycosylated and processed during secretion by a yeast host. Secreted hGM–CSF, but not mGM–CSF was significantly O–glycosylated. Human GM–CSF was expressed mainly as a highly N–glycosylated form, while 50% of mGM–CSF was unglycosylated, or core–glycosylated. The Asn–X–Thr and Asn–X–Ser sequences of hGM–CSF were equally efficient sites for N–glycosylation, which enhanced the secretion efficiency of hGM–CSF, but not of mGM–CSF. N–terminal analysis of secreted hGM–CSF revealed a novel processing site following the pre–region of the MFα1/hGM–CSF precursor and in addition, suggested partial N–terminal degradation by the STE13–encoded dipeptidase. GM–CSFs secreted by yeast and by animal cells had identical in vitro biological activities.

The anti-inflammatory action of glucocorticoids has been attributed to the induction of a group of phospholipase A2inhibitory proteins, collectively called lipocortin. These proteins are thought to control the biosynthesis of the potent mediators of inflammation, prostaglandins and leukotrienes, by inhibiting the release of their common precursor, arachidonic acid, a process that requires phospholipase A2hydrolysis of phospholipids1–11. Lipocortin-like proteins have been isolated from various cell types, including monocytes3, neutrophils4and renal medullary9cell preparations. The predominant active form is a protein with an apparent relative molecular mass (Mr) of 40,000 (40K). These partially purified preparations of lipocortin mimic the effect of steroids, and mediate anti-inflammatory activity in various in vivo model systems3,5,11. Using amino-acid sequence information obtained from purified rat lipocortin, we have now cloned human lipocortin complementary DNA and expressed the gene in Escherichia coli. Our studies confirm that lipocortin is a potent inhibitor of phospholipase A2activity.

The manner in which a membrane protein is anchored to the lipid bilayer may have a profound influence on its function. Most cell surface membrane proteins are anchored by a membrane-spanning segment(s) of the polypeptide chain, but another type of anchor has been described for several proteins: a phosphatidyl inositol glycan moiety, attached to the protein C terminus1,2. This type of linkage has been identified on membrane proteins involved in adhesion3and transmembrane signalling4,5and could be important in the execution of these functions. We report here that an immunologically important adhesion glycoprotein, lymphocyte function-associated antigen 3 (LFA-3), can be anchored to the membrane by both types of mechanism. These two distinct cell-surface forms of LFA-3 are derived from different biosynthetic precursors. The existence of a phosphatidyl-inositol-linked and a transmembrane anchored form of LFA-3 has important implications for adhesion and transmembrane signalling by LFA-3.