Posttranslational N-glycosylation takes place during the normal processing of human coagulation factor VII

N-glycosylation is normally a cotranslational process that occurs during translocation of the nascent protein to the endoplasmic reticulum. In the present study, however, we demonstrate posttranslational N-glycosylation of recombinant human coagulation factor VII (FVII) in CHO-K1 and 293A cells. Human FVII has two N-glycosylation sites (N145 and N322). Pulse-chase labeled intracellular FVII migrated as two bands corresponding to FVII with one and two N-glycans, respectively. N-glycosidase treatment converted both of these band into a single band, which comigrated with mutated FVII without N-glycans. Immediately after pulse, most labeled intracellular FVII had one N-glycan, but during a 1-h chase, the vast majority was processed into FVII with two N-glycans, demonstrating posttranslational N-glycosylation of FVII. Pulse-chase analysis of N-glycosylation site knockout mutants demonstrated cotranslational glycosylation of N145 but primarily or exclusively posttranslational glycosylation of N322. The posttranslational N-glycosylation appeared to take place in the same time frame as the folding of nascent FVII into a secretion-competent conformation, indicating a link between the two processes. We propose that the cotranslational conformation(s) of FVII are unfavorable for glycosylation at N332, whereas a more favorable conformation is obtained during the posttranslational folding. This is the first documentation of posttranslational N-glycosylation of a non-modified protein in mammalian cells with an intact N-glycosylation machinery. Thus, the present study demonstrates that posttranslational N-glycosylation can be a part of the normal processing of glycoproteins.     

Temporal variations of coagulation factor VII activity in mice are influenced by lighting regime

It was recently reported that the circadian clock machinery controls plasma levels of factor (F) VII, the serine protease triggering blood coagulation. Here, by exploiting the mouse model, this study showed that variations of photoperiod (i.e., winter or summer conditions or simulated chronic jetlag conditions) have a strong impact on plasma FVII activity levels. Under conditions mimicking summer or winter photoperiods, FVII activity showed a clear 24 h rhythmicity. Interestingly, mean daily FVII activity levels were significantly reduced in mice exposed to summer photoperiods. Behavioral activity rhythms under both photoperiods were synchronized to LD cycles, and the amount of activity per 24 h was comparable. The authors also investigated the influence of chronic jetlag (CJL) on the FVII activity rhythms, which can be easily mimicked in mice through continuous abrupt shifts in the lighting schedule. The exposure of mice to simulated CJL of either consecutive westward or consecutive westward and eastward flights for 15 days did not abolish the behavioral activity rhythms but was associated with a period significantly different from 24 h. Intriguingly, both types of CJL exerted a strong influence on FVII activity rhythms, which were virtually suppressed. Moreover, the mean daily FVII activity was significantly lower in the CJL than in the winter photoperiod condition. Taken together, these findings in mice provide novel insights into the modulation of FVII activity levels, which might have implications for human pathophysiology.     

Mass spectrometric characterization of mitochondrial complex I NDUFA10 variants

In the present study, we have used a combination of 2-DE and MS to isolate and characterize two variants of the mitochondrial complex I subunit NDUFA10 from Wistar rat brain. Extensive MS/MS analysis revealed that a D/N substitution at position 120 resulting from a 353A/G transition in the coding gene is the biochemical difference between the two most abundant NDUFA10 isoforms. Moreover, 33 modifications of distinct chemical nature targeting 59 specific residues were found to be common to the acidic and basic forms. Positions C67, H149 and H322 of NDUFA10 were specially targeted by different modifications suggesting the high reactivity of these residues and their potential implication in the regulation of the protein function. Together with nonenzymatic modifications that can form in the sample isolation and workup steps, such as oxidation of methionine, tryptophan, cysteine and histidine, we describe amino acid variants of unknown chemical structure that must be further characterized, as well as accumulation of R, K and H methylations and probably K acetylations at the C-terminal region that might play a role in the control of NDUFA10 activity according to similar mechanisms to those described for histones.     

Mass spectrometric analysis of histone posttranslational modifications

The uses of tandem and Fourier transform mass spectrometric methodologies for assignment of the posttranslational sites and occupancies of histones and their isoforms is described employing several illustrative examples. A comparison of information that can be obtained from intact protein sequencing and proteolytic digestion is presented.     

A rapid mass spectrometric strategy for the characterization of N- and O-glycan chains in the diagnosis of defects in glycan biosynthesis

Glycosylation of proteins is a very complex process which involves numerous factors such as enzymes or transporters. A defect in one of these factors in glycan biosynthetic pathways leads to dramatic disorders named congenital disorders of glycosylation (CDG). CDG can affect the biosynthesis of not only protein N-glycans but also O-glycans. The structural analysis of glycans on serum glycoproteins is essential to solving the defect. For this reason, we propose in this paper a strategy for the simultaneous characterization of both N- and O-glycan chains isolated from the serum glycoproteins. The serum (20 microL) is used for the characterization of N-glycans which are released by enzymatic digestion with PNGase F. O-glycans are chemically released by reductive elimination from whole serum glycoproteins using 10 microL of the serum. Using strategies based on mass spectrometric analysis, the structures of N- and O-glycan chains are defined. These strategies were applied on the sera from one patient with CDG type IIa, and one patient with a mild form of congenital disorder of glycosylation type II (CDG-II) that is caused by a deficiency in the Cog1 subunit of the complex.     

Characterization of N- and O-linked glycosylation of recombinant human bile salt-stimulated lipase secreted by Pichia pastoris

Recombinant human bile salt-stimulated lipase (hBSSL) was expressed in and secreted by Pichia pastoris, an organism exploited for the large-scale production of recombinant (glyco)proteins by bioprocessing technology. The 76.3-kDa glycoprotein was associated with 75-80 Man and a small amount of GlcNAc. hBSSL has one N-glycosylation site at Asn187, which was 38-40% occupied with a Man(10)GlcNAc(2) structure defined previously in Pichia as the oligosaccharide-lipid form of Man(9)GlcNAc(2) trimmed of the middle-arm terminal alpha 1,2-Man and elongated with Man alpha 1,2Man alpha 1,6-disaccharide attached to the lower-arm core alpha 1,3-Man (Trimble et al. [1991], J. Biol. Chem., 266, 22807-22817). The C-terminal 192 residues of hBSSL contain 16 Pro-rich 11-amino-acid repeats, which include 32 Ser/Thr residues as potential O-glycosylation sites. Using hBSSL as a platform to study Pichia's O-glycosylation capabilities, we found that nearly all of these sites were occupied by mannose-containing O-glycans, whose structures, after beta-elimination and purification, were assigned by (1)H NMR and, in some cases, by linkage-specific exoglycosidases and methylation analysis. The most abundant O-glycan was alpha 1,2-mannobiitol (55%), followed by alpha 1,2-mannotriitol (16%) and mannitol (10%) and a lesser amount was alpha 1,2-mannotetraitol. Unexpectedly, Man(5) and Man(6) O-glycans were present, which had the structure Man beta 1,2Man beta 1,2Man alpha 1,2(Man alpha 1,2)(1,2)mannitol. Also a small amount of a phosphorylated Man(6) O-glycan was characterized by MALDI-TOF MS postsource decay analysis as having the reducing-end mannitol disubstituted with a glycosidically linked phosphorylated Man and an unbranched Man(4) polymer elongated from a different mannitol carbon. This is the first report of the synthesis of beta-Man- and phosphate-containing O-linked constituents on glycoproteins synthesized by P. pastoris.     

Site‐specific N‐glycosylation analysis of human factor XI: Identification of a noncanonical NXC glycosite

Human factor XI (hFXI) is a 160‐kDa disulphide‐linked homodimer zymogen involved in the coagulation cascade. Its deficiency results in bleeding diathesis referred to as hemophilia C. hFXI bears five N‐glycosylation consensus sites per monomer, N₇₂, N₁₀₈, N₃₃₅ on the heavy chain and N₄₃₂, N₄₇₃ on the light chain. This study reports the first in‐depth glycosylation analysis of hFXI based on advanced MS approaches. Hydrophilic interaction LC and MS characterization and quantification of the N‐glycans showed that the two major forms are complex biantennary mono‐α2,6‐sialylated (A₂S₁, 20%) and bis‐α2,6‐sialylated structures (A₂S₂, 66%). Minor triantennary structures (A₃S₃F, ～1.5%; A₃S₃, ～2%) were also identified. MS analyses of intact hFXI revealed full occupation of two of the three heavy‐chain glycosites and almost full‐site occupancy of the light chain. Analysis of hFXI glycopeptides by LC‐MS/MS enabled site‐specific glycan profiling and occupancy. It was evidenced that N₃₃₅ was not glycosylated and that N₇₂ and N₁₀₈ were fully occupied, whereas N₄₃₂ and N₄₇₃ were occupied at about 92 and 95%, respectively. We also identified a new glycosite of the noncanonical format NXC at N₁₄₅, occupied at around 5%. These data provide valuable structural information useful to understand the potential roles of N‐glycosylation on hFXI function and could serve as a structural reference.     

The influence of different glycosylation patterns on factor VII biological activity

In this study the bioactivity of three differently glycosylated blood coagulation factor VII (FVII) variants (human plasma FVII, recombinant human FVII produced in CHO and BHK cell cultures) were analyzed and compared. Surface plasmon resonance studies of FVII interaction with soluble and full length TF together with FVII autoactivation assays revealed that BHK-derived FVII has the highest bioactivity, while human plasma and CHO-derived FVII showed very similar bioactivity. The affinity of FVII variants to TF correlates with FVII autoactivation rates – the higher the affinity, the faster the autoactivation rate.     

Mass spectrometric measurements of the apolipoproteins of bovine (Bos taurus) HDL

As is the case in most mammals, high density lipoproteins (HDL) also comprise the major group of lipid carriers that circulate in bovine (Bos taurus) blood. As a continuation of our proteogenomic studies of mammalian apolipoproteins, we have obtained molecular masses for several of the apolipoproteins associated with bovine HDL. The major apolipoprotein on the HDL surface is apoA-I, but other apolipoproteins were also detected. Using electrospray-ionization mass spectrometry (ESI-MS), we report on values for apolipoproteins, A-I, proA-I and A-II, as well as post-translationally modified apoA-I. Analyses of tryptic fragments did reveal the presence of apoA-IV and apoC-III. However, in contrast to our previous studies of other mammalian HDL, we did not detect apoC-I. Interestingly, examination of the current assembly for the bovine genome does not show any evidence for an apoC-I gene.     

Mass spectrometric protein structure characterization reveals cause of migration differences of haptoglobin alpha chains in two-dimensional gel electrophoresis

Haptoglobin belongs to the major constituents of plasma and acts as hemoglobin-binding and acute-phase protein. Due to the occurrence of three major allelic variants and further structural modifications, the alpha chains of haptoglobin form varying spot patterns in two-dimensional gel electrophoresis (2-DE) gels, which is generally observed in differential proteome analyses using plasma or related body fluids of humans. In the present study plasma samples from 10 donors of initially unknown haptoglobin phenotype were separated by 2-DE and tryptic digests of excised haptoglobin alpha chain spots were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and MALDI-quadrupole ion trap TOF-MS. Haptoglobin alpha1S, alpha1F, as well as alpha2 chains were found to occur each with at least three structurally differing protein species: (i) the unmodified form, which corresponds to the sequence database entries; (ii) derivatives, in which asparagine at position five is deamidated to aspartic acid; and (iii) derivatives with an additional C-terminal arginine residue. These structural variants account for the most commonly observed spot patterns of haptoglobin alpha chains in Coomassie-stained gels. Additionally, a minor derivative of the haptoglobin alpha2 chain carrying both modifications, deamidation at position five and the C-terminal arginine residue, was identified. Theoretical pI values of the characterized structural variants are, consistent with their observed migration in the 2-DE gels.     

Mass spectrometric identification of novel posttranslational modification sites in Huntingtin

Huntington's disease (HD) is caused by a CAG triplet repeat expansion in exon 1 of the Huntingtin (Htt) gene, encoding an abnormal expanded polyglutamine (polyQ) tract that confers toxicity to the mutant Htt (mHtt) protein. Recent data suggest that posttranslational modifications of mHtt modulate its cytotoxicity. To further understand the cytotoxic mechanisms of mHtt, we have generated HEK293 cell models stably expressing Strep- and FLAG-tagged Htt containing either 19Q (wild-type Htt), 55Q (mHtt), or 94Q (mHtt) repeats. Following tandem affinity purification, the tagged Htt and associated proteins were subjected to tandem mass spectrometry or 2D nano-LC tandem mass spectrometry and several novel modification sites of mHtt containing 55Q or 94Q were identified. These were phosphorylation sites located at Ser431 and Ser432, and ubiquitination site located at Lys444. The two phosphorylation sites were confirmed by Western blot analysis using phosphorylation site-specific antibodies. In addition, prevention of phosphorylation at the two serine sites altered mHtt toxicity and accumulation. These modifications of mHtt may provide novel therapeutic targets for effective treatment of the disorder.     

Mass spectrometric quantification of asparagine synthetase in circulating leukemia cells from acute lymphoblastic leukemia patients

The appearance of asparaginase-resistant acute lymphoblastic leukemia (ALL) in transformed cell lines has been correlated with increased expression of asparagine synthetase (ASNS). Recent measurements using mRNA-based assays have raised doubts, however, as to the importance of ASNS protein in the cellular mechanisms that confer drug resistance upon the leukemic cells. Studies aimed at determining the concentration of ASNS protein in human leukemias are therefore needed to resolve this issue. A mass spectrometry (MS)-based procedure is presented for the direct quantification of ASNS protein concentration in complex sample mixtures. This assay is able to distinguish samples from transformed cell lines that express ASNS over a wide dynamic range of concentration. Importantly, this method directly detects ASNS protein, the functional entity that may be synthesizing sufficient asparagine to render leukemia cells resistant to asparaginase-treatment. We also report the successful use of this MS method, which has lower limits of detection and quantification of 30 and 100 attomoles, respectively, for the first direct measurements of ASNS protein concentrations in four patient blast samples.     

人凝血酶原复合物生产过程中凝血因子活化分析

目的通过比较以组分Ⅲ沉淀和血浆为原料制备人凝血酶原复合物(Prothrombin complex concentrates,PCC)过程中凝血因子活化情况,为选择最适PCC制备原料提供数据支持。方法分别对以组分Ⅲ沉淀和血浆为原料制备PCC过程中中间品的活化的凝血因子活性和人凝血酶活性两个项目进行检定,分析凝血因子的活化情况。观察以组分Ⅲ沉淀为原料制备PCC过程中添加肝素能否抑制PCC中凝血因子的活化。结果以组分Ⅲ沉淀为原料制备的PCC中间品活化的凝血因子活性和人凝血酶活性两个项目均不合格。以组分Ⅲ沉淀为原料制备PCC生产过程中添加肝素后,PCC中间品的活化的凝血因子活性和人凝血酶活性均不合格。以血浆为原料制备的PCC中间品活化的凝血因子活性和人凝血酶活性两个项目均合格。结论组分Ⅲ沉淀为原料制备PCC会增加凝血因子活化的风险,新鲜冰冻血浆可作为制备PCC的原料。     

Mass spectrometric quantitation of C-reactive protein using labeled tryptic peptides

Given the extensive efforts applied toward proteomics and research in biomarkers, methods for the simultaneous measurement of proteins, peptides, metabolic intermediates, hormones, etc. in a complex sample may be required in the foreseeable future. Assays based on mass spectrometric detection may be suitable for meeting the demands of such complex samples with sensitivity and specificity. An analytical method for the quantitation of C-reactive protein (CRP), a well-known marker of inflammation, is described. Exact quantities of two synthetic (13)C-labeled CRP tryptic peptides were added as internal standards directly to the sample prior to chemical treatment, trypsinization, and liquid chromatography/mass spectrometry quantitation. C-reactive protein levels based on isotopic response ratios were measured. Intact C-reactive protein was spiked into blank rat urine for chemical and enzymatic treatment, producing linear response ratios of labeled to unlabeled peptides. For rigorous quantitation, standard curves, and quality control samples were prepared in rat urine with highly purified labeled and unlabeled peptides over the 50 pg-5 ng/muL concentration range. Using the same chemical and enzymatic treatment used for digestion of intact CRP, data from these samples demonstrated excellent analytical performance. The method was successfully applied toward the quantitation of urinary C-reactive protein from a study of drug-induced nephrotoxicity.     

Purification of human factor VII utilizing O-(diethylaminoethyl)-Sephadex and Sulfopropyl-Sephadex chromatography

A simple procedure for the large scale purification of unactivated human factor VII is described. The initial steps, common to prior purification methods, include adsorption onto barium citrate, ammonium sulfate fractionation, and DEAE-Sephadex chromatography. Factor VII is isolated in pure unactivated form by one additional step, Sulfopropyl-Sephadex chromatography. Ten liters of plasma yields 1.3 mg of protein representing approximately 30% recovery.     

Temporal Variations of Coagulation Factor VII Activity in Mice Are Influenced by Lighting Regime

It was recently reported that the circadian clock machinery controls plasma levels of factor (F) VII, the serine protease triggering blood coagulation. Here, by exploiting the mouse model, this study showed that variations of photoperiod (i.e., winter or summer conditions or simulated chronic jetlag conditions) have a strong impact on plasma FVII activity levels. Under conditions mimicking summer or winter photoperiods, FVII activity showed a clear 24 h rhythmicity. Interestingly, mean daily FVII activity levels were significantly reduced in mice exposed to summer photoperiods. Behavioral activity rhythms under both photoperiods were synchronized to LD cycles, and the amount of activity per 24 h was comparable. The authors also investigated the influence of chronic jetlag (CJL) on the FVII activity rhythms, which can be easily mimicked in mice through continuous abrupt shifts in the lighting schedule. The exposure of mice to simulated CJL of either consecutive westward or consecutive westward and eastward flights for 15 days did not abolish the behavioral activity rhythms but was associated with a period significantly different from 24 h. Intriguingly, both types of CJL exerted a strong influence on FVII activity rhythms, which were virtually suppressed. Moreover, the mean daily FVII activity was significantly lower in the CJL than in the winter photoperiod condition. Taken together, these findings in mice provide novel insights into the modulation of FVII activity levels, which might have implications for human pathophysiology.     

Random analysis of amino acid pairs sensitive to variants in human coagulation factor IX precursor

In this study, we used a random approach to determine which amino acid pairs in human coagulation factor IX precursor are more sensitive to its 99 variants. The results show that the randomly unpredictable amino acid pairs are more sensitive to variants.     

Rational combinatorial chemistry-based selection,synthesis and evaluation of an affinity adsorbent for recombinant human clotting factor VII

The selection, synthesis and chromatographic evaluation of a synthetic affinity adsorbent for human recombinant factor VIIa is described. The requirement for a metal ion-dependent immunoadsorbent step in the purification of the recombinant human clotting factor, FVIIa, has been obviated by using the X-ray crystallographic structure of the complex of tissue factor (TF) and Factor VIIa and has directed our combinatorial approach to select, synthesise and evaluate a rationally-selected affinity adsorbent from a limited library of putative ligands. The selected and optimised ligand comprises a triazine scaffold bis-substituted with 3-aminobenzoic acid and has been shown to bind selectively to FVIIa in a Ca(2+)-dependent manner. The adsorbent purifies FVIIa to almost identical purity (>99%), yield (99%), activation/degradation profile and impurity content (approximately 1000 ppm) as the current immunoadsorption process, while displaying a 10-fold higher static capacity and substantially higher reusability and durability.     

Mass spectrometric analysis of single identified neurons of an insect

The combination of retrograde labelling with dextran-tetramethylrhodamine and MALDI-TOF mass spectrometry was used to analyse for the first time the peptidome of a series of morphologically identified single neurosecretory cells of an insect. Eight postero-lateral cells of the metathoracic ganglion of the American cockroach, Periplaneta americana, were used to demonstrate that: (1) the complete dissection procedure can be documented and (2) the mass spectrometric analysis of the dissected somata results in highly reproducible mass spectra. In total, 21 FMRFamide-related peptides were detected in each of the postero-lateral cells which release their neurosecretions via thoracic perisympathetic organs. Direct analysis of these neurohemal organs confirmed the co-storage of FMRFamide-related peptides. Two additional abundant peptides from thoracic perisympathetic organs which were not detectable in the postero-lateral cells were characterized using ESI-Q-TOF MS/MS. De novo sequencing yielded two related peptides (FERL/IEamides) without any similarity with known peptide families of insects.