The ␥ subunit of the Na,K-ATPase is a member of the FXYD family of type 2 transmembrane proteins that probably function as regulators of ion transport. Rat ␥ is present primarily in the kidney as two main splice variants, ␥ a and ␥ b , which differ only at their extracellular N termini (TELSANH and MDRWYL, respectively; Kuster, B., Shainskaya, A., Pu, H. X., Goldshleger, R., Blostein, R., Mann, M., and Karlish, S. J. D. (2000) J. Biol. Chem. 275, 18441-18446). Expression in cultured cells indicates that both variants affect catalytic properties, without a detectable difference between ␥ a and ␥ b . At least two singular effects are seen, irrespective of whether the variants are expressed in HeLa or rat ␣1-transfected HeLa cells, i.e. (i) an increase in apparent affinity for ATP, probably secondary to a left shift in E 1 7 E 2 conformational equilibrium and (ii) an increase in K ؉ antagonism of cytoplasmic Na ؉ activation. Antibodies against the C terminus common to both variants (anti-␥) abrogate the first effect but not the second. In contrast, ␥ a and ␥ b show differences in their localization along the kidney tubule. Using anti-␥ (C-terminal) and antibodies to the rat ␣ subunit as well as antibodies to identify cell types, double immunofluorescence showed ␥ in the basolateral membrane of several tubular segments. Highest expression is in the medullary portion of the thick ascending limb (TAL), which contains both ␥ a and ␥ b . In fact, TAL is the only positive tubular segment in the medulla. In the cortex, most tubules express ␥ but at lower levels. Antibodies specific for ␥ a and ␥ b showed differences in their cortical location; ␥ a is specific for cells in the macula densa and principal cells of the cortical collecting duct but not cortical TAL. In contrast, ␥ b but not ␥ a is present in the cortical TAL only. Thus, the importance of ␥ a and ␥ b may be related to their partially overlapping but distinct expression patterns and tissue-specific functions of the pump that these serve.A small membrane protein, ␥, first described over 20 years ago in purified kidney Na,K-ATPase preparations (1, 2) associates, in approximately equimolar amounts, with the ␣ and ␤ subunits (3, 4). Molecular cloning of the ␥ subunits of rat, mouse, cow, and sheep indicated a molecular weight of ϳ6500 (5). Cloning and sequencing of the human (6) and Xenopus laevis (7) ␥ subunits have also been reported. Comparison of sequences shows ϳ75% homology among ␥ subunits of the aforementioned different species but is much higher (93%) for only mammalian sequences. Further structural analysis has shown that ␥ comprises a single transmembrane domain and has an N terminus-out, C terminus-in topology (7,8). In addition, two major forms have been recently identified at the molecular level as described below.On SDS-polyacrylamide gel electrophoresis, the ␥ subunit runs as a doublet (apparent molecular masses of ϳ8 and ϳ9 kDa) (5,8), and a doublet is observed following expression in tissue culture cells (8, 9) and in in vitro expression in the pre...

Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.

The two variants of the ␥ subunit of the rat renal sodium pump, ␥ a and ␥ b , have similar effects on the Na,K-ATPase. Both increase the affinity for ATP due to a shift in the enzyme's E 1 7 E 2 conformational equilibrium toward E 1 . In addition, both increase K ؉ antagonism of cytoplasmic Na ؉ activation. To gain insight into the structural basis for these distinct effects, extramembranous N-terminal and C-terminal mutants of ␥ were expressed in rat ␣1-transfected HeLa cells. At the N terminus, the variant-distinct region was deleted (␥N⌬7) or replaced by alanine residues (␥N7A). At the C terminus, four (␥ a C⌬4) or ten (␥ a C⌬10) residues were deleted. None of these mutations abrogates the K ؉ /Na ؉ antagonism as evidenced in a similar increase in K Na seen at high (100 mM) K ؉ concentration. In contrast, the C-terminal as well as N-terminal deletions (␥N⌬7, ␥ a C⌬4, and

Background:The role of GOLPH3 in mammalian glycosylation is not well studied. Results: GOLPH3 binds to and controls the Golgi localization of POMGnT1. Conclusion: GOLPH3 regulates the glycosylation of ␣-dystroglycan by anchoring POMGnT1 at the Golgi. Significance: The result provides a further understanding of the role of GOLPH3 in mediating the Golgi localization of glycosyltransferases in mammalian cells.