Increased myocardial Rab GTPase expression: a consequence and cause of cardiomyopathy.

The Ras-like Rab GTPases regulate vesicle transport in endocytosis and exocytosis. We found that cardiac Rabs1, 4, and 6 are upregulated in a dilated cardiomyopathy model overexpressing beta(2)-adrenergic receptors. To determine if increased Rab GTPase expression can contribute to cardiomyopathy, we transgenically overexpressed in mouse hearts prototypical Rab1a, the small G protein that regulates vesicle transport from endoplasmic reticulum to and through Golgi. In multiple independent mouse lines, Rab1a overexpression caused cardiac hypertrophy that progressed in a time- and transgene dose-dependent manner to heart failure. Isolated cardiac myocytes were hypertrophied and exhibited contractile depression with impaired calcium reuptake. Ultrastructural analysis revealed enlarged Golgi stacks and increased transitional vesicles in ventricular myocytes, with increased secretory atrial natriuretic peptide granules and degenerative myelin figures in atrial myocytes; immunogold studies localized Rab1a to these abnormal vesicular structures. A survey of hypertrophy signaling molecules revealed increased protein kinase C (PKC) alpha and delta, and confocal microscopy showed abnormal subcellular distribution of PKCalpha in Rab1a transgenics. These results indicate that increased expression of Rab1 GTPase in myocardium distorts subcellular localization of proteins and is sufficient to cause cardiac hypertrophy and failure.

Validation of a peptide mapping method for a therapeutic monoclonal antibody: what could we possibly learn about a method we have run 100 times?

Peptide mapping is a key analytical method for studying the primary structure of proteins. The sensitivity of the peptide map to even the smallest change in the covalent structure of the protein makes it a valuable 'finger-print' for identity testing and process monitoring. We recently conducted a full method validation study of an optimised reverse-phase high-performance liquid chromatography (RP-HPLC) tryptic map of a therapeutic anti-CD4 IgG1 monoclonal antibody. We have used this method routinely for over 1 year to support bioprocess development and test production lots for clinical trials. Herein we summarize the precision and ruggedness of the testing procedure and the main findings with respect to 'coverage of amino acid sequence' and limits-of-detection for various hypothetical structural variants. We also describe, in more detail, two unanticipated insights into the method gained from the validation study. The first of these is a potentially troublesome side-product arising during the reduction/alkylation step. Once the cause of this side-product was identified, it was easily prevented. We also report on subtle changes to the peptide map upon extended storage of the digest in the autosampler. These findings helped us to develop a 'robust' method for implementation in a quality control laboratory.

Identification and characterization of glycosylation sites in human serum clusterin.

Clusterin is a ubiquitous, heterodimeric glycoprotein with multiple possible functions that are likely influenced by glycosylation. Identification of oligosaccharide attachment sites and structural characterization of oligosaccharides in human serum clusterin has been performed by mass spectrometry and Edman degradation. Matrix-assisted laser desorption ionization mass spectrometry revealed two molecular weight species of holoclusterin (58,505 +/- 250 and 63,507 +/- 200). Mass spectrometry also revealed molecular heterogeneity associated with both the alpha and beta subunits of clusterin, consistent with the presence of multiple glycoforms. The data indicate that clusterin contains 17-27% carbohydrate by weight, the alpha subunit contains 0-30% carbohydrate and the beta subunit contains 27-30% carbohydrate. Liquid chromatography electrospray mass spectrometry with stepped collision energy scanning was used to selectively identify and preparatively fractionate tryptic glycopeptides. Edman sequence analysis was then used to confirm the identities of the glycopeptides and to define the attachment sites within each peptide. A total of six N-linked glycosylation sites were identified, three in the alpha subunit (alpha 64N, alpha 81N, alpha 123N) and three in the beta subunit (beta 64N, beta 127N, and beta 147N). Seven different possible types of oligosaccharide structures were identified by mass including: a monosialobiantennary structure, bisialobiantennary structures without or with one fucose, trisialotriantennary structures without or with one fucose, and possibly a trisialotriantennary structure with two fucose and/or a tetrasialotriantennary structure. Site beta 64N exhibited the least glycosylation diversity, with two detected types of oligosaccharides, and site beta 147N exhibited the greatest diversity, with five or six detected types of oligosaccharides. Overall, the most abundant glycoforms detected were bisialobiantennary without fucose and the least abundant were monosialobiantennary, trisialotriantennary with two fucose and/or tetrasialotriantennary. Clusterin peptides accounting for 99% of the primary structure were identified from analysis of the isolated alpha and beta subunits, including all Ser- and Thr-containing peptides. No evidence was found for the presence of O-linked or sulfated oligosaccharides. The results provide a molecular basis for developing a better understanding of clusterin structure-function relationships and the role clusterin glycosylation plays in physiological function.

1,25-dihydroxyvitamin D3 modulates phosphorylation of serine 205 in the human vitamin D receptor: site-directed mutagenesis of this residue promotes alternative phosphorylation.

The vitamin D receptor (VDR) from a variety of animal species is a hormone-modulated substrate for phosphorylation in vivo. In this report, we utilize an expression vector to produce recombinant human VDR (hVDR) in 1,25-dihydroxyvitamin D3-treated COS-1 cells. Immunoprecipitation of the phosphorylated hVDR followed by gel purification and phosphoamino acid analysis revealed modification exclusively on one or more serine residues, consistent with previous studies of the VDR in other species. To identify the region of phosphorylation, immunoprecipitated and gel-purified hVDR from COS-1 cells was first mixed with purified hVDR isolated to homogeneity from Saccharomyces cerevisiae and then digested with trypsin or V8 protease, and the peptides were resolved on HPLC. The single phosphate-containing peptides were recovered and subjected to amino acid sequence analysis, revealing the modification to reside in a region extending from residue 171 to residue 206 common to both the tryptic- and the V8 protease-derived peptides. Sequential cleavage of similar VDR mixtures using trypsin and then CNBr, alpha-chymotrypsin, or thermolysin demonstrated an amino-terminal boundary of the phosphorylated peptide at 202. Selective manual Edman degradation of phosphorylated peptides beginning at 171, 195, and 200 revealed phosphate release only at serine 205. This peptide contained an average of 8-fold less radioactive phosphate in the absence of prior treatment of the culture cells with 1,25(OH)2D3. Site-directed modification of VDR serine 205 to alanine, aspartate, or glutamate each led to fully functional proteins when assessed in a transactivation assay using several VDRE-linked natural promoters. Unexpectedly, evaluation of the serine 205 to alanine hVDR mutant revealed that this protein continued to be phosphorylated in a hormone-dependent manner on an alternative site. These studies show directly that hVDR serine residue 205, a consensus site for casein kinase II, is modified in vivo in response to hormone.

Positive regulation of the vitamin D receptor by its cognate ligand in heterologous expression systems.

Hormonal vitamin D3 is a major regulator of calcium metabolism and is involved in basic cellular processes, such as those of proliferation and differentiation. These actions are mediated via an intracellular vitamin D3 receptor (VDR), which is a member of the evergrowing steroid hormone receptor superfamily. The interaction between the vitamin D3 ligand and its receptor is thought to be through a classic steroid hormone mechanism. It is notable, however, that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] has also been documented as an agent that directly up-regulates endogenous VDR in both intact animals and cultured cells. In this report, we confirm that the levels of recombinantly expressed VDR produced in transiently transfected COS-1 cells also increase several-fold when the cells are treated with 1,25-(OH)2D3. Additionally, we show that a similar pattern is exhibited in a Saccharomyces cerevisiae expression system. This indicates that the mechanism for VDR up-regulation is conserved in both yeast and mammalian cells. Our results show that up-regulation by 1,25-(OH)2D3 is specific to the VDR, and that 1,25-(OH)2D3 does not affect other expressed receptor proteins, such as those for estrogen and progesterone. Finally, we demonstrate that the mechanism of up-regulation apparently occurs at the level of the protein and is most likely due to altered stability of the occupied receptor. Our observations lead us to propose that in addition to the classically viewed role of hormone in receptor activation, 1,25-(OH)2D3 may serve to amplify signal response via homologous up-regulation.