13C15N: glucagon-based novel isotope dilution mass spectrometry method for measurement of glucagon metabolism in humans.

BACKGROUND: Glucagon serves as an important regulatory hormone for regulating blood glucose concentration with tight feedback control exerted by insulin and glucose. There are critical gaps in our understanding of glucagon kinetics, pancreatic α cell function and intra-islet feedback network that are disrupted in type 1 diabetes. This is important for translational research applications of evolving dual-hormone (insulin + glucagon) closed-loop artificial pancreas algorithms and their usage in type 1 diabetes. Thus, it is important to accurately measure glucagon kinetics in vivo and to develop robust models of glucose-insulin-glucagon interplay that could inform next generation of artificial pancreas algorithms. METHODS: Here, we describe the administration of novel 13C15N heavy isotope-containing glucagon tracers-FF glucagon [(Phe 6 13C9,15N; Phe 22 13C9,15N)] and FFLA glucagon [(Phe 6 13C9,15N; Phe 22 13C9,15N; Leu 14 13C6,15N; Ala 19 13C3)] followed by anti-glucagon antibody-based enrichment and LC-MS/MS based-targeted assays using high-resolution mass spectrometry to determine levels of infused glucagon in plasma samples. The optimized assay results were applied for measurement of glucagon turnover in subjects with and without type 1 diabetes infused with isotopically labeled glucagon tracers. RESULTS: The limit of quantitation was found to be 1.56 pg/ml using stable isotope-labeled glucagon as an internal standard. Intra and inter-assay variability was < 6% and < 16%, respectively, for FF glucagon while it was < 5% and < 23%, respectively, for FFLA glucagon. Further, we carried out a novel isotope dilution technique using glucagon tracers for studying glucagon kinetics in type 1 diabetes. CONCLUSIONS: The methods described in this study for simultaneous detection and quantitation of glucagon tracers have clinical utility for investigating glucagon kinetics in vivo in humans.

Serum levels of DNAJB9 are elevated in fibrillary glomerulonephritis patients.

Fibrillary glomerulonephritis (FGN) is a rare glomerular disease. Kidney biopsy is required to establish the diagnosis. Recent studies have identified abundant glomerular deposition of DNAJB9 as a unique histological marker of FGN. We developed an immunoprecipitation-based multiple reaction monitoring method to measure serum levels of DNAJB9. We detected a 4-fold higher abundance of serum DNAJB9 in FGN patients when compared to controls, including patients with other glomerular diseases. Serum DNAJB9 levels were also negatively associated with estimated glomerular filtration rate in patients with FGN. Serum DNAJB9 levels accurately predicted FGN with moderate sensitivity (67%) and with high specificity (98%) and positive and negative predictive value (89% and 95%, respectively). A receiver operating curve analysis demonstrated an AUC of 0.958. These results suggest that serum levels of DNAJB9 could be a valuable marker to predict FGN, with the potential to complement kidney biopsy for the diagnosis of FGN.

Phosphatidylserine Stimulates Ceramide 1-Phosphate (C1P) Intermembrane Transfer by C1P Transfer Proteins.

Genetic models for studying localized cell suicide that halt the spread of pathogen infection and immune response activation in plants include Arabidopsis accelerated-cell-death 11 mutant (acd11). In this mutant, sphingolipid homeostasis is disrupted via depletion of ACD11, a lipid transfer protein that is specific for ceramide 1-phosphate (C1P) and phyto-C1P. The C1P binding site in ACD11 and in human ceramide-1-phosphate transfer protein (CPTP) is surrounded by cationic residues. Here, we investigated the functional regulation of ACD11 and CPTP by anionic phosphoglycerides and found that 1-palmitoyl-2-oleoyl-phosphatidic acid or 1-palmitoyl-2-oleoyl-phosphatidylglycerol (≤15 mol %) in C1P source vesicles depressed C1P intermembrane transfer. By contrast, replacement with 1-palmitoyl-2-oleoyl-phosphatidylserine stimulated C1P transfer by ACD11 and CPTP. Notably, "soluble" phosphatidylserine (dihexanoyl-phosphatidylserine) failed to stimulate C1P transfer. Also, none of the anionic phosphoglycerides affected transfer action by human glycolipid lipid transfer protein (GLTP), which is glycolipid-specific and has few cationic residues near its glycolipid binding site. These findings provide the first evidence for a potential phosphoglyceride headgroup-specific regulatory interaction site(s) existing on the surface of any GLTP-fold and delineate new differences between GLTP superfamily members that are specific for C1P versus glycolipid.

Mutational Constraints on Local Unfolding Inhibit the Rheological Adaptation of von Willebrand Factor.

Unusually large von Willebrand factor (VWF), the first responder to vascular injury in primary hemostasis, is designed to capture platelets under the high shear stress of rheological blood flow. In type 2M von Willebrand disease, two rare mutations (G1324A and G1324S) within the platelet GPIbα binding interface of the VWF A1 domain impair the hemostatic function of VWF. We investigate structural and conformational effects of these mutations on the A1 domain's efficacy to bind collagen and adhere platelets under shear flow. These mutations enhance the thermodynamic stability, reduce the rate of unfolding, and enhance the A1 domain's resistance to limited proteolysis. Collagen binding affinity is not significantly affected indicating that the primary stabilizing effect of these mutations is to diminish the platelet binding efficiency under shear flow. The enhanced stability stems from the steric consequences of adding a side chain (G1324A) and additionally a hydrogen bond (G1324S) to His(1322) across the ß2-ß3 hairpin in the GPIbα binding interface, which restrains the conformational degrees of freedom and the overall flexibility of the native state. These studies reveal a novel rheological strategy in which the incorporation of a single glycine within the GPIbα binding interface of normal VWF enhances the probability of local unfolding that enables the A1 domain to conformationally adapt to shear flow while maintaining its overall native structure.

Conformational Changes in the Endosomal Sorting Complex Required for the Transport III Subunit Ist1 Lead to Distinct Modes of ATPase Vps4 Regulation.

Intralumenal vesicle formation of the multivesicular body is a critical step in the delivery of endocytic cargoes to the lysosome for degradation. Endosomal sorting complex required for transport III (ESCRT-III) subunits polymerize on endosomal membranes to facilitate membrane budding away from the cytoplasm to generate these intralumenal vesicles. The ATPase Vps4 remodels and disassembles ESCRT-III, but the manner in which Vps4 activity is coordinated with ESCRT-III function remains unclear. Ist1 is structurally homologous to ESCRT-III subunits and has been reported to inhibit Vps4 function despite the presence of a microtubule-interacting and trafficking domain-interacting motif (MIM) capable of stimulating Vps4 in the context of other ESCRT-III subunits. Here we report that Ist1 inhibition of Vps4 ATPase activity involves two elements in Ist1: the MIM itself and a surface containing a conserved ELYC sequence. In contrast, the MIM interaction, in concert with a more open conformation of the Ist1 core, resulted in stimulation of Vps4. Addition of the ESCRT-III subunit binding partner of Ist1, Did2, also converted Ist1 from an inhibitor to a stimulator of Vps4 ATPase activity. Finally, distinct regulation of Vps4 by Ist1 corresponded with altered ESCRT-III disassembly in vitro. Together, these data support a model in which Ist1-Did2 interactions during ESCRT-III polymerization coordinate Vps4 activity with the timing of ESCRT-III disassembly.

Enzymatic discovery of a HER-2/neu epitope that generates cross-reactive T cells.

Patients with HER-2/neu-expressing breast cancer remain at risk for relapse following standard therapy. Vaccines targeting HER-2/neu to prevent relapse are in various phases of clinical testing. Many vaccines incorporate the HER-2/neu HLA-A2-binding peptide p369-377 (KIFGSLAFL), because it has been shown that CTLs specific for this epitope can directly kill HER-2/neu-overexpressing breast cancer cells. Thus, understanding how tumors process this epitope may be important for identifying those patients who would benefit from immunization. Proteasome preparations were used to determine if p369-377 was processed from larger HER-2/neu-derived fragments. HPLC, mass spectrometry, cytotoxicity assays, IFN-γ ELISPOT, and human breast cancer cell lines were used to assess the proteolytic fragments. Processing of p369-377 was not detected by purified 20S proteasome and immunoproteasome, indicating that tumor cells may not be capable of processing this Ag from the HER-2/neu protein and presenting it in the context of HLA class I. Instead, we show that other extracellular domain HER-2/neu peptide sequences are consistently processed by the proteasomes. One of these sequences, p373-382 (SLAFLPESFD), bound HLA-A2 stronger than did p369-377. CTLs specific for p373-382 recognized both p373-382 and p369-377 complexed with HLA-A2. CTLs specific for p373-382 also killed human breast cancer cell lines at higher levels than did CTLs specific for p369-377. Conversely, CTLs specific for p369-377 recognized p373-382. Peptide p373-382 is a candidate epitope for breast cancer vaccines, as it is processed by proteasomes and binds HLA-A2.

Relative quantification: characterization of bias, variability and fold changes in mass spectrometry data from iTRAQ-labeled peptides.

Shotgun proteomics via mass spectrometry (MS) is a powerful technology for biomarker discovery that has the potential to lead to noninvasive disease screening mechanisms. Successful application of MS-based proteomics technologies for biomarker discovery requires accurate expectations of bias, reproducibility, variance, and the true detectable differences in platforms chosen for analyses. Characterization of the variability inherent in MS assays is vital and should affect interpretation of measurements of observed differences in biological samples. Here we describe observed biases, variance structure, and the ability to detect known differences in spike-in data sets for which true relative abundance among defined samples were known and were subsequently measured with the iTRAQ technology on two MS platforms. Global biases were observed within these data sets. Measured variability was a function of mean abundance. Fold changes were biased toward the null and variance of a fold change was a function of protein mass and abundance. The information presented herein will be valuable for experimental design and analysis of the resulting data.

Structural determination and tryptophan fluorescence of heterokaryon incompatibility C2 protein (HET-C2), a fungal glycolipid transfer protein (GLTP), provide novel insights into glycolipid specificity and membrane interaction by the GLTP fold.

HET-C2 is a fungal protein that transfers glycosphingolipids between membranes and has limited sequence homology with human glycolipid transfer protein (GLTP). The human GLTP fold is unique among lipid binding/transfer proteins, defining the GLTP superfamily. Herein, GLTP fold formation by HET-C2, its glycolipid transfer specificity, and the functional role(s) of its two Trp residues have been investigated. X-ray diffraction (1.9 A) revealed a GLTP fold with all key sugar headgroup recognition residues (Asp(66), Asn(70), Lys(73), Trp(109), and His(147)) conserved and properly oriented for glycolipid binding. Far-UV CD showed secondary structure dominated by alpha-helices and a cooperative thermal unfolding transition of 49 degrees C, features consistent with a GLTP fold. Environmentally induced optical activity of Trp/Tyr/Phe (2:4:12) detected by near-UV CD was unaffected by membranes containing glycolipid but was slightly altered by membranes lacking glycolipid. Trp fluorescence was maximal at approximately 355 nm and accessible to aqueous quenchers, indicating free exposure to the aqueous milieu and consistent with surface localization of the two Trps. Interaction with membranes lacking glycolipid triggered significant decreases in Trp emission intensity but lesser than decreases induced by membranes containing glycolipid. Binding of glycolipid (confirmed by electrospray injection mass spectrometry) resulted in a blue-shifted emission wavelength maximum (approximately 6 nm) permitting determination of binding affinities. The unique positioning of Trp(208) at the HET-C2 C terminus revealed membrane-induced conformational changes that precede glycolipid uptake, whereas key differences in residues of the sugar headgroup recognition center accounted for altered glycolipid specificity and suggested evolutionary adaptation for the simpler glycosphingolipid compositions of filamentous fungi.

Simultaneous phenotyping and quantification of α-1-antitrypsin by liquid chromatography-tandem mass spectrometry.

BACKGROUND: α-1-Antitrypsin (A1AT) deficiency results from a genetic disorder at 2 common loci. Diagnosis requires quantification of A1AT and subsequent identification of the specific variant. The current algorithm of laboratory testing for the diagnosis of A1AT deficiency uses a combination of quantification (nephelometry), genotyping, and/or phenotyping. We developed a multiple reaction monitoring liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of A1AT and identification of the 2 most common deficiency alleles present in 95% of the patients with A1AT deficiency. METHODS: Serum samples (n = 40) were digested with trypsin, and appropriate ¹³C/¹5N-labeled standard peptides were added. We performed LC-MS/MS analysis with a 0.5- by 150-mm C18 column and H2O:acetonitrile:n-propanol:formic acid (A:98:1:1:0.2 and B:10:80:10:0.2; flow 12 µL/min) mobile phase in positive ion mode on a TSQ Quantum triple quadrupole MS system. We measured the A1AT concentration by comparison to a calibration curve and determined the phenotype by the presence or absence of variant peptides. We compared the results to the current phenotyping assay by isoelectric focusing (IEF) and the immunonephelometry quantitative assay. RESULTS: For A1AT allele detection, in 39 of 40 samples the LC-MS/MS results were identical to those obtained by IEF gel electrophoresis. The single discrepant result was rerun by IEF at a lower dilution, and the results were in concordance. The A1AT quantification by LC-MS/MS also compared favorably with nephelometry. CONCLUSIONS: The LC-MS/MS method correlates well with current phenotyping and nephelometric assays and has the potential to improve the laboratory diagnosis of genetic A1AT deficiency.

Recognition of fungal protease activities induces cellular activation and eosinophil-derived neurotoxin release in human eosinophils.

Eosinophils are multifunctional leukocytes implicated in the pathogenesis of asthma and in immunity to certain organisms. Associations between exposure to an environmental fungus, such as Alternaria, and asthma have been recognized clinically. Protease-activated receptors (PARs) are G protein-coupled receptors that are cleaved and activated by serine proteases, but their roles in innate immunity remain unknown. We previously found that human eosinophils respond vigorously to Alternaria organisms and to the secretory product(s) of Alternaria with eosinophils releasing their proinflammatory mediators. In this study, we investigated the roles of protease(s) produced by Alternaria and of PARs expressed on eosinophils in their immune responses against fungal organisms. We found that Alternaria alternata produces aspartate protease(s) and that human peripheral blood eosinophils degranulate in response to the cell-free extract of A. alternata. Eosinophils showed an increased intracellular calcium concentration in response to Alternaria that was desensitized by peptide and protease ligands for PAR-2 and inhibited by a PAR-2 antagonistic peptide. Alternaria-derived aspartate protease(s) cleaved PAR-2 to expose neo-ligands; these neo-ligands activated eosinophil degranulation in the absence of proteases. Finally, treatment of Alternaria extract with aspartate protease inhibitors, which are conventionally used for HIV-1 and other microbes, attenuated the eosinophils' responses to Alternaria. Thus, fungal aspartate protease and eosinophil PAR-2 appear critical for the eosinophils' innate immune response to certain fungi, suggesting a novel mechanism for pathologic inflammation in asthma and for host-pathogen interaction.