Human Plasma Proteins Research Articles

Abstract 1132: Regulation of CT120 gene as a new target of c-myc in head and neck squamous cell carcinoma (HNSCC)

Abstract HNSCC is the sixth most common form of cancer and represents the third common cause of cancer-related deaths worldwide. Therefore, there is a need to understand the molecular mechanisms involved in the pathogenesis of HNSCC. The c-myc protein belongs to the myc family of transcription factors and plays a fundamental role in cell cycle progression, apoptosis and cellular transformation. In the human genome, the MYC gene is located on chromosome 8 and its protein product regulates expression of 15% of all genes through binding to the Enhancer box (E-box) sequences. An E-box is a DNA sequence found in the promoter regions of eukaryotic genes that acts as a protein binding site to regulate gene expression. The CANNTG consensus sequence of the E-box is known as the canonical E-boxes. There are several other non-canonical E-box motifs such as the GATGTG, CATGCG, CACGCG, CACGAG, CGCGAG and CAACGTG sequences. Amplification of the MYC gene is observed in many different types of cancers including HNSCC. CT120 is a novel gene which encodes a human trans-membrane plasma protein. It has been shown that up-regulation of CT120 is associated with lung and head and neck carcinogenesis. In this study we investigated the promoter region of the CT120 gene and identified 6 E-boxes. To test whether c-myc binds to these E-boxes we performed chromatin immunoprecipitation (ChIP) assays. We demonstrated that c-myc binds strongly to the E-boxes 2, 3 and 4. It is well known that the expression rates of the genes are under the control of epigenetic modifications. Methylation of the CpG islands which are found in the promoter regions is one of the most studied epigenetic modifications. We identified two putative CpG islands in the promoter of the CT120 gene. To investigate the possible epigenetic regulation of CT120 and the effect of the promoter methylation to the binding of c-myc, we also investigated the methylation levels of the two islands in the CT120 promoter by MS-PCR. We observed a slight decrease (3.1%) in the methylation level of the first CpG-rich region. In 56% (28/50) of the tumor tissues methylation was decreased compared to the non-cancerous tissues. However, there was no statistically significant correlation between the promoter methylation and CT120 mRNA level. Depending on these data we investigated the expression levels of the c-myc and CT120 in the 50 HNSCC and normal tissue samples by qRT-PCR and observed a direct correlation between the overexpression of the c-myc and CT120 genes. Overexpression of CT120 and c-myc were observed in 29 (58%) and 34 (68%) of the tumors, respectively, compared to non-cancerous tissues. A total of 24 (48%) patients out of 50 showed a concurrent pattern of either up- or down regulation. Our results indicate that the CT120 gene is a target of c-myc and that both proteins may participate in the progression of HNSCC. Citation Format: Onur Baykara, Elif Baltaci, Betul Seyhan, Nejat Dalay, Nur Buyru. Regulation of CT120 gene as a new target of c-myc in head and neck squamous cell carcinoma (HNSCC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1132.

Serial estimation of serum albumin and its role in traumatic brain injury patients

Background: Serum albumin is routinely measured; cheap and easily available test even in remote areas laboratories. Albumin is the major protein of human plasma and one of the negative acute phase reactants reported to fall as a component of metabolic response to injury or infection, independent of the nutritional status. Even though this has also been noted in patients with head injury and its overall significance with respect to neurological outcome following head injury are yet to be established.Aims and Objectives: To assess the role of serum albumin in outcome of head injury patient.Material and Methods: This is a prospective case control study. Total 200 cases of traumatic brain injury [152 male and 48 female] were studied in NRS Medical College, Kolkata from September 2012 to October 2013. Serum albumin estimation, along with clinical and radiological data was collected. Serum albumin was compared between among various subgroups and outcome was assessed by Glasgow Coma Scale at 6 months.Results: In present study 86(43%) patients having hypoalbuminemia (< 3.5gm/dl) and falling trend in admission serum albumin levels from day 1 to 10th day with decrement in GCS was statistically highly significant (p < 0.001) as hypoalbuminemia was noted in 68%, 39.65 and 33.43% patients with severe, moderate and mild head injury respectively. The present study was showed that female TBI patients having less no. of hypoalbuminemia compare to male as 77.7%, 45.4%, 31.4% male and 42.8%, 28.5%, 27.2% female having hypoalbuminemia (< 3.5gm/dl) in severe, moderate and mild head injury respectively and statistically significant difference in severe and moderate group.Conclusion: The serial estimation of serum albumin in traumatic brain injury patients provides the treating doctor an insight in the prognosis of the patient so that they can be managed aggressively.Asian Journal of Medical Sciences Vol.7(4) 2016 31-38

Differential compartmentalization of Streptococcus pyogenes virulence factors and host protein binding properties as a mechanism for host adaptation

Streptococcus pyogenes is an important human pathogen responsible for substantial morbidity and mortality worldwide. Although S. pyogenes is a strictly human pathogen with no other known animal reservoir, several murine infection models exist to explore different aspects of the bacterial pathogenesis. Inoculating mice with wild-type S. pyogenes strains can result in the generation of new bacterial phenotypes that are hypervirulent compared to the original inoculum. In this study, we used a serial mass spectrometry based proteomics strategy to investigate if these hypervirulent strains have an altered distribution of virulence proteins across the intracellular, surface associated and secreted bacterial compartments and if any change in compartmentalization can alter the protein-protein interaction network between bacteria and host proteins. Quantitative analysis of the S. pyogenes surface and secreted proteomes revealed that animal passaged strains are associated with significantly higher amount of virulence factors on the bacterial surface and in the media. This altered virulence factor compartmentalization results in increased binding of several mouse plasma proteins to the bacterial surface, a trend that was consistent for mouse plasma from several different mouse strains. In general, both the wild-type strain and animal passaged strain were capable of binding high amounts of human plasma proteins. However, compared to the non-passaged strains, the animal passaged strains displayed an increased ability to bind mouse plasma proteins, in particular for M protein binders, indicating that the increased affinity for mouse blood plasma proteins is a consequence of host adaptation of this pathogen to a new host. In conclusion, plotting the total amount of virulence factors against the total amount of plasma proteins associated to the bacterial surface could clearly separate out animal passaged strains from wild type strains indicating a virulence model that could predict the virulence of a S. pyogenes strain in mice and which could be used to identify key aspects of this bacteria’s pathogenesis.

DETERMINATION OF FUNDAMENTAL PHYSICAL PARAMETERS OF BLOOD SERUM PROTEINS FOR DEVELOPMENT THE METHODS FOR CANCER DIAGNOSIS

Актуальность. Для медицинской диагностики фундаментальной проблемой является исследование воздействия патологического состояния человека на белки плазмы и сыворотки крови. В качестве диагностических параметров предлагается использовать физические молекулярно-динамические характеристики белков, которые существенно изменяются при развитии болезни. Цель – проведение фундаментальных исследований молекулярных свойств белков сыворотки крови (их подвижность, межмолекулярные взаимодействия, агрегация – образование наноразмерных белковых кластеров, изменение поверхностного заряда и др.) для развития методики диагностики онкологических заболеваний, направленной на создание экспресс-метода на базе лазерного светорассеяния и флуоресценции. Материал и методы. Для определения динамических параметров белков использованы методы лазерного светорассеяния. Результаты. Проведены сравнительные исследования модельных систем и нативных образцов сыворотки крови для диагностики новообразований и рассмотрена возможность применения оптических параметров водных растворов сыворотки крови для создания методов диагностики онкологических заболеваний. Заключение. Методы светорассеяния дают достаточную ин- формацию об изменении статических и динамических свойств белков сыворотки крови при патологии.

Solvatochromic fluorescent probes for recognition of human serum albumin in aqueous solution: Insights into structure-property relationship

Human serum albumin (HSA) as the most abundant protein in human blood plasma, serves many physiological functions. The dysregulation of HSA in serum or in urine is associated with various diseases, such as cirrhosis of liver, multiple myeloma, and cardiovascular disease. Therefore, to quantify HSA in body fluids with high selectivity and sensitivity is of great significance for disease diagnosis and preventive medicine. We herein developed a series of amide-functionalized flavonoids probes, 1–3, for recognition of human serum albumin. All flavonoids could be easily prepared by a Claisen-Schmidt condensation and Algar-Flynn-Oyamada reaction, and showed positive solvatochromism on their dual emissions. The chemical structure of flavonoids played an important role on their HSA-sensing abilities. Among three probes, the compound 1 showed the highest sensitivity, the remarkable selectivity, and the quantitive response for HSA in aqueous solution. Together with its high tolerance of environmental pH, anti-interference properties, and time-insensitivity, thus it provides a promising sensing method for HSA.

Study of interactions between blood plasma proteins and poly(butyl cyanoacrylate) drug nanocarriers by surface plasmon resonance

Investigation and novel data on the interactions between blood plasma proteins and polysorbate 80-coated poly(butyl cyanoacrylate) (PBCA) drug nanocarriers determined by surface plasmon resonance (SPR) are reported. For that purpose, human blood plasma proteins (albumin, immunoglobulin G and fibrinogen) were immobilized on the surface of SPR sensorchips and the kinetics of nanoparticle interaction (adsorption/desorption) with the chip surface was studied at 37°C at pH 7.4. The proposed methodology was found suitable for measurement of interactions of both drug-free and drug-loaded nanocarriers with blood plasma proteins and allowed quantitative evaluation of both rate and equilibrium constants of interactions. Presented results demonstrated that SPR can be considered as a promising technique for real time investigation of both kinetic and equilibrium parameters of the nanoparticle-protein interactions, which could be of importance for the development of drug nanocarrier systems and their biomedical evaluation and application.

Decellularized GGTA1-KO pig heart valves do not bind preformed human xenoantibodies.

Pre-clinical and clinical data have unequivocally demonstrated the usefulness of decellularized heart valve (HV) matrices implanted for HV replacement therapy. However, human donor valves applicable for decellularization are in short supply, which prompts the search for suitable alternatives, such as porcine grafts. Since decellularization might be insufficient to remove all xenoantigens, we analysed the interaction of human preformed antibodies with decellularized porcine HV in vitro to assess potential immune reactions upon implantation. Detergent-decellularized pulmonary HV from German Landrace wild-type (wt) or α1,3-galactosyltransferase knockout (GGTA1-KO) pigs were investigated by inhibition ELISA and GSL I-B4 staining to localize and quantify matrix-bound αGal epitopes, which represent the most prominent xenoantigen. Additionally, preformed human xenoantibodies were affinity purified by perfusing porcine kidneys. Binding of purified human antibodies to decellularized HV was investigated by inhibition ELISA. Furthermore, binding of human plasma proteins to decellularized matrices was determined by western blot. Decellularized human pulmonary artery served as controls. Decellularization of wt HV led to a reduction of αGal epitopes by 70%. Residual epitopes were associated with the subendothelial extracellular matrix. As expected, no αGal epitopes were found on decellularized GGTA1-KO matrix. The strongest binding of preformed human anti-pig antibodies was found on wt matrices, whereas GGTA1-KO matrices bound similar or even fewer xenoantibodies than human controls. These results demonstrate the suitability of GGTA1-KO pigs as donors for decellularized heart valves for human patients. Besides the presence of αGal antibodies on decellularized heart valves, no further preformed xenoantibodies against porcine matrix were detected in tested human sera.

A comparative analysis of serum albumin from different species to determine a natural source of albumin that might be useful for human therapy

Abstract Objectives Serum albumin (SA) is one of the most abundant proteins in human plasma and performs functions for protein transport, scavenges for free radicals, and regulates osmotic pressure. SA has been used for therapy in humans with several diseases including hypovolemia, hypoalbuminemia, and chronic liver disease. However, the most appropriate source of albumin for medical applications remains unclear, especially in the case of viral infections, which complicates finding viable donors. Substitution of human serum albumin (HSA) with albumin from other natural sources is a viable alternative. Therefore, we elucidated the similarity in character, structure and evolutionary relationship among serum albumin isolated from seven different species. Methods We compared the sequence, structure, and properties of SA from different species using an in-silico approach. Results These data suggested that SA has sequence polymorphism that clusters based on closely related-species. However, these polymorphisms do not change the three-dimensional structure of the protein; this may serve to maintain its function as a transporter. The Gallus gallus albumin has the lowest number of the epitopes that closely resemble HSA. Conclusion This study is crucial in providing explicit information about the structural similarity of albumin isolated from other species compared to HSA. The Gallus gallus SA might be used as a primary natural source of albumin where warranted for human therapy.

Nitroimidazole carboxamides as antiparasitic agents targeting Giardia lamblia, Entamoeba histolytica and Trichomonas vaginalis

Diarrhoeal diseases caused by the intestinal parasites Giardia lamblia and Entamoeba histolytica constitute a major global health burden. Nitroimidazoles are first-line drugs for the treatment of giardiasis and amebiasis, with metronidazole 1 being the most commonly used drug worldwide. However, treatment failures in giardiasis occur in up to 20% of cases and development of resistance to metronidazole is of concern. We have re-examined ‘old’ nitroimidazoles as a foundation for the systematic development of next-generation derivatives. Using this approach, derivatisation of the nitroimidazole carboxamide scaffold provided improved antiparasitic agents. Thirty-three novel nitroimidazole carboxamides were synthesised and evaluated for activity against G. lamblia and E. histolytica. Several of the new compounds exhibited potent activity against G. lamblia strains, including metronidazole-resistant strains of G. lamblia (EC50 = 0.1–2.5 μM cf. metronidazole EC50 = 6.1–18 μM). Other compounds showed improved activity against E. histolytica (EC50 = 1.7–5.1 μM cf. metronidazole EC50 = 5.0 μM), potent activity against Trichomonas vaginalis (EC50 = 0.6–1.4 μM cf. metronidazole EC50 = 0.8 μM) and moderate activity against the intestinal bacterial pathogen Clostridium difficile (0.5–2 μg/mL, cf. metronidazole = 0.5 μg/mL). The new compounds had low toxicity against mammalian kidney and liver cells (CC50 > 100 μM), and selected antiparasitic hits were assessed for human plasma protein binding and metabolic stability in liver microsomes to demonstrate their therapeutic potential.

Where Did the Linker-Payload Go? A Quantitative Investigation on the Destination of the Released Linker-Payload from an Antibody-Drug Conjugate with a Maleimide Linker in Plasma.

The reactive thiol of cysteine is often used for coupling maleimide-containing linker-payloads to antibodies resulting in the generation of antibody drug conjugates (ADCs). Currently, a numbers of ADCs in drug development are made by coupling a linker-payload to native or engineered cysteine residues on the antibody. An ADC conjugated via hinge-cysteines to an auristatin payload was used as a model in this study to understand the impact of the maleimide linkers on ADC stability. The payload was conjugated to trastuzumab by a protease-cleavable linker, maleimido-caproyl-valine-citruline-p-amino-benzyloxy carbonyl (mcVC-PABC). In plasma stability assays, when the ADC (Trastuzumab-mcVC-PABC-Auristatin-0101) was incubated with plasma over a 144-h time-course, a discrepancy was observed between the measured released free payload concentration and the measured loss of drug-to-antibody ratio (DAR), as measured by liquid chromatography-mass spectrometry (LC-MS). We found that an enzymatic release of payload from ADC-depleted human plasma at 144 h was able to account for almost 100% of the DAR loss. Intact protein mass analysis showed that at the 144 h time point, the mass of the major protein in ADC-depleted human plasma had an additional 1347 Da over the native albumin extracted from human plasma, exactly matching the mass of the linker-payload. In addition, protein gel electrophoresis showed that there was only one enriched protein in the 144 h ADC-depleted and antipayload immunoprecipitated plasma sample, as compared to the 0 h plasma immunoprecipitated sample, and the mass of this enriched protein was slightly heavier than the mass of serum albumin. Furthermore, the albumin adduct was also identified in 96 h and 168 h postdose in vivo cynomolgus monkey plasma. These results strongly suggest that the majority of the deconjugated mc-VC-PABC-auristatin ultimately is transferred to serum albumin, forming a long-lived albumin-linker-payload adduct. To our knowledge, this is the first report quantitatively characterizing the extent of linker-payload transfer to serum albumin and the first clear example of in vivo formation of an albumin-linker-payload adduct.

Tetrabromobisphenol A Is an Efficient Stabilizer of the Transthyretin Tetramer.

Amyloid formation of the human plasma protein transthyretin (TTR) is associated with several human disorders, including familial amyloidotic polyneuropathy (FAP) and senile systemic amyloidosis. Dissociation of TTR’s native tetrameric assembly is the rate-limiting step in the conversion into amyloid, and this feature presents an avenue for intervention because binding of an appropriate ligand to the thyroxin hormone binding sites of TTR stabilizes the native tetrameric assembly and impairs conversion into amyloid. The desired features for an effective TTR stabilizer include high affinity for TTR, high selectivity in the presence of other proteins, no adverse side effects at the effective concentrations, and a long half-life in the body. In this study we show that the commonly used flame retardant tetrabromobisphenol A (TBBPA) efficiently stabilizes the tetrameric structure of TTR. The X-ray crystal structure shows TBBPA binding in the thyroxine binding pocket with bromines occupying two of the three halogen binding sites. Interestingly, TBBPA binds TTR with an extremely high selectivity in human plasma, and the effect is equal to the recently approved drug tafamidis and better than diflunisal, both of which have shown therapeutic effects against FAP. TBBPA consequently present an interesting scaffold for drug design. Its absorption, metabolism, and potential side-effects are discussed.

Polyphenolic–polysaccharide conjugates from plants of Rosaceae/Asteraceae family as potential radioprotectors

Polyphenolic–polysaccharide macromolecular, water-soluble glycoconjugates, isolated from the selected medicinal plants of Rosaceae/Asteraceae family: from leaves of Fragaria vesca L., Rubus plicatus Whe. et N. E., and from flowering parts of Sanguisorba officinalis L., and Erigeron canadensis L., were investigated for their ability to protect proteins and lipids of human plasma against γ-radiation-induced oxidative damage. Treatment of plasma with plant conjugates (6, 30, 150μg/ml) prior exposure to 100Gy radiation resulted in a significant inhibition of lipid peroxidation, evaluated by TBARS levels; conjugates isolated from E. canadensis and R. plicatus and a reference flavonoid quercetin showed similar high potential (approx. 70% inhibition, at 6μg/ml). The conjugates prevented radiation-induced oxidation of protein thiols and significantly improved plasma total antioxidant capacity, estimated with Ellman’s reagent and ABTS.+ assay, respectively.The results demonstrate by the first time a significant radioprotective capability of the polyphenolic-polysaccharide conjugates isolated from E. canadensis, R. plicatus, S. officinalis and to the less extent from F. vesca. The abilities of these substances to inhibit radiation-induced lipid peroxidation and thiol oxidation in plasma seems to be mediated, but not limited to ROS scavenging activity.

Artificial Neural Network Analysis of Pharmacokinetic and Toxicity Properties of Lead Molecules for Dengue Fever, Tuberculosis and Malaria.

Poor pharmacokinetic and toxicity profiles are major reasons for the low rate of advancing lead drug candidates into efficacy studies. The In-silico prediction of primary pharmacokinetic and toxicity properties in the drug discovery and development process can be used as guidance in the design of candidates. In-silico parameters can also be used to choose suitable compounds for in-vivo testing thereby reducing the number of animals used in experiments. At the Novartis Institute for Tropical Diseases, a data set has been curated from in-house measurements in the disease areas of Dengue, Tuberculosis and Malaria. Volume of distribution, half-life, total in-vivo clearance, in-vitro human plasma protein binding and in-vivo oral bioavailability have been measured for molecules in the lead optimization stage in each of these three disease areas. Data for the inhibition of the hERG channel using the radio ligand binding dofetilide assay was determined for a set of 300 molecules in these therapeutic areas. Based on this data, Artificial Neural Networks were used to construct In-silico models for each of the properties listed above that can be used to prioritize candidates for lead optimization and to assist in selecting promising molecules for in-vivo pharmacokinetic studies.

Thermal Stability of Human Serum Albumin: the Dependence on the Protein Concentration, Scan Rate, and the Presence of Fatty Acids and Low-Weight Molecular Ligands

Human serum albumin (HSA) is the most abundant protein in human plasma with ascribed ligand binding and transport properties. Design of this molecule provides a variety of binding sites for many physiological important ligands, including a wide variety of drugs. In this work, the dependence of the thermal denaturation of HSA on the protein concentration, scan rate, and the presence of fatty acids and low-molecular weight drugs (warfarin, ibuprofen, hypericin, emodin) have been thoroughly investigated by means of differential scanning calorimetry (DSC). Our results show that the presence of fatty acids in HSA significantly stabilizes the structure of this protein. Further, DSC curves exhibit a significant dependence on the protein concentration in both fatty acid containing and fatty acids free HSA. From the linear dependence of 1/Tm on albumin concentration follows that HSA tends to form dimers during denaturation process. The obtained DSC data show only a slight increase of Tm and ΔHcal with a scan rate increase. Interactions of the studied low-molecular weight drugs with HSA lead to a significant enhancement of the protein thermal stability (higher temperature and enthalpy of the denaturation in comparison with those thermodynamic parameters determined for HSA alone). However, in the presence of emodin at high concentration ratios (emodin:HSA>10:1), a decrease of the stabilization effect is observed. This is probably caused by the ability of emodin to bind both folded and unfolded states of HSA.AcknowledgementsThis work has been supported by the contract APVV-0242-11, the project CELIM (316310) funded by 7 FP EU, and by the EU Structural Funds (ITMS 26240120040 and ITMS 26220220107(50%)).

Human Serum Albumin-[Ru(Phen)3]2+ Complex Formation Studied by Optical Spectroscopies

Assessment of the tissue oxygenation is an important factor allowing to analyze various processes depending on the presence of oxygen. However, the oxygen sensitive probes can interact with proteins or lipids (depending on the hydrophobic character of the probe) what can interfere with the precise determination of the oxygen concentration. Dichlorotris(1,10-phenanthroline)-ruthenium(II)hydrate ([Ru(Phen)3]2+) is a water soluble non-phototoxic compound serving as a sensor of molecular oxygen. This type of heterocyclic ligands can also be used for antitumor, antibacterial and antiviral purposes. Thus, its interaction with carrier protein such as serum albumin is of high importance since this complex formation has a substantial influence on the in vivo distribution of the sensor.In this work, we have investigated the mechanism of [Ru(Phen)3]2+ interaction with human serum albumin (HSA), the most abundant protein in human plasma, by using different optical spectroscopy techniques: UV-visible absorption, fluorescence and Raman spectroscopies. The obtained experimental data suggest only weak interaction of [Ru(Phen)3]2+ with HSA (Kd= 64 μM). Further, Raman spectroscopy and surface-enhanced Raman spectroscopy allowed us to register so-called fingerprint vibrational spectra of both molecules as well as to detect small changes in the polypeptide backbone conformations induced by the [Ru(Phen)3]2+-molecule binding.AcknowledgementsThis work was funded by the contract APVV-0242-11, by the EU 7FP grant CELIM 316310 and by the project of the EU Structural Funds (ITMS 26240120040).

Structural and Functional Study on the Interaction of Human Plasminogen and Protein H from Haemophilus Influenzae Type F

Haemophilus influenzae serotype f (Hif) resides in human respiratory tract, is an emerging pathogen causes invasive diseases. Recently, we annotated the whole genome of an invasive Hif isolate and showed that Hif simultaneously interacts with factor H and vitronectin by using the surface protein H (PH). Here, upon screening with various human plasma proteins, we found that PH can also interact with plasminogen, an important coagulation regulator of the blood. Plasminogen bound at a bacterial surface can be converted into plasmin and degrades complement factors such as C3b and C5 and thus suppresses the host innate immunity. In order to study the structural interaction between PH and human plasminogen, we cloned the lph gene encoding the protein PH in pETM30 vector and optimized the expression and purification of untagged recombinant PH. The dissociation constant (Kd) of the interaction between recombinant PH and plasminogen was 2.7 μM as measured by Biolayer interferometry and Microscale thermophoresis. We demonstrate that Hif can bind plasminogen, which is converted to plasmin that degrades complement factor C3b, C5 and blood clotting factor fibrinogen. We are now crystallizing PH, and the PH-plasminogen complex. Our study will spot light on deeper understanding of Hif virulence.

Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins

Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1 mucin-type O-glycans (T-antigen). The developed workflow allows the identification and characterization of the major population of the human blood plasma O-glycoproteome and our results provide new insights, which can help to unravel structure-function relationships. The data were deposited to ProteomeXchange PXD003270.

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Array-Based Online Two Dimensional Liquid Chromatography System Applied to Effective Depletion of High-Abundance Proteins in Human Plasma.

In this work, an array-based online two-dimensional liquid chromatography (2D-LC) system was constructed for protein separation and effective depletion of high-abundance proteins in human plasma. This system employed a strong anion exchange column in the first dimension and eight reversed-phase liquid chromatographic columns in the second dimension. All the protein components in the first dimension were enriched on the trapping columns, simultaneously back-flushed and concurrently separated in the second dimension. LC eluents were then collected on 96-well plates for further analysis. Compared with common 2D-LC system, this system showed an 8-fold increase in throughput and convenient utilization of stop-flow mode for sample separation. The RSD of retention time and peak area were separately below 0.51% and 8%. Recovery rates of four standard proteins were all above 95%. This array-based 2D-LC system was subsequently applied to the analysis of proteins in human plasma. The eluents containing high-abundance proteins were rapidly located according to the results of bicinchoninic acid assay. In all, with the effective depletion of 84 high-abundance proteins, a total of 1332 proteins were identified through our system. The dynamic range of the identified protein concentrations covered 9 orders of magnitude, ranging from 41 g/L level for HSA down to 0.01 ng/mL level for the low-abundance proteins.

Identification of Human Sulfotransferases Involved in Lorcaserin N-Sulfamate Formation.

Lorcaserin [(R)-8-chloro-1-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine] hydrochloride hemihydrate, a selective serotonin 5-hydroxytryptamine (5-HT) 5-HT(2C) receptor agonist, is approved by the U.S. Food and Drug Administration for chronic weight management. Lorcaserin is primarily cleared by metabolism, which involves multiple enzyme systems with various metabolic pathways in humans. The major circulating metabolite is lorcaserin N-sulfamate. Both human liver and renal cytosols catalyze the formation of lorcaserin N-sulfamate, where the liver cytosol showed a higher catalytic efficiency than renal cytosol. Human sulfotransferases (SULTs) SULT1A1, SULT1A2, SULT1E1, and SULT2A1 are involved in the formation of lorcaserin N-sulfamate. The catalytic efficiency of these SULTs for lorcaserin N-sulfamate formation is widely variable, and among the SULT isoforms SULT1A1 was the most efficient. The order of intrinsic clearance for lorcaserin N-sulfamate is SULT1A1 > SULT2A1 > SULT1A2 > SULT1E1. Inhibitory effects of lorcaserin N-sulfamate on major human cytochrome P450 (P450) enzymes were not observed or minimal. Lorcaserin N-sulfamate binds to human plasma protein with high affinity (i.e., >99%). Thus, despite being the major circulating metabolite, the level of free lorcaserin N-sulfamate would be minimal at a lorcaserin therapeutic dose and unlikely be sufficient to cause drug-drug interactions. Considering its formation kinetic parameters, high plasma protein binding affinity, minimal P450 inhibition or induction potential, and stability, the potential for metabolic drug-drug interaction or toxicological effects of lorcaserin N-sulfamate is remote in a normal patient population.