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1.
Diabetes ; 69(4): 760-770, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31974145

RESUMO

Long-term hyperglycemia in patients with diabetes leads to human serum albumin (HSA) glycation, which may impair HSA function as a transport protein and affect the therapeutic efficacy of anticoagulants in patients with diabetes. In this study, a novel mass spectrometry approach was developed to reveal the differences in the profiles of HSA glycation sites between patients with diabetes and healthy subjects. K199 was the glycation site most significantly changed in patients with diabetes, contributing to different interactions of glycated HSA and normal HSA with two types of anticoagulant drugs, heparin and warfarin. An in vitro experiment showed that the binding affinity to warfarin became stronger when HSA was glycated, while HSA binding to heparin was not significantly influenced by glycation. A pharmacokinetic study showed a decreased level of free warfarin in the plasma of diabetic rats. A preliminary retrospective clinical study also revealed that there was a statistically significant difference in the anticoagulant efficacy between patients with diabetes and patients without diabetes who had been treated with warfarin. Our work suggests that larger studies are needed to provide additional specific guidance for patients with diabetes when they are administered anticoagulant drugs or drugs for treating other chronic diseases.

2.
Carbohydr Polym ; 231: 115695, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31888803

RESUMO

Building blocks characterization is a significant approach for understanding the molecular structure of heparin and its derivatives. Nitrous acid (HONO) depolymerization of heparin generates oligosaccharides that maintain the epimerization conformation on C5 of the uronic acids, reflecting the authentic structure of the parental chain. HONO treatment at pH 1.5 selectively cleaves the bond between N-sulfated glucosamine and hexuronic acid, resulting mainly disaccharides, as well as tetra-, tri-, and mono-saccharides. The tetrasaccharides are derived from the structure of N-acetylated domains while tri-, and mono-saccharides are derived from the reducing or the non-reducing end of the heparin chain. The resulted oligosaccharides were separated and analyzed using a UHPLC-HILIC/WAX-MS method. We succeeded in the identification of 19 tetrasaccharides, 19 trisaccharides and 4 monosaccharides species, majority of which is structurally characterized. By comparing the theoretical possibilities and actual occurrence of the well-characterized tetrasaccharides, we demonstrated that the biosynthesis of heparin is a systematic process.


Assuntos
Heparina/química , Estrutura Molecular , Ácido Nitroso/química , Oligossacarídeos/química , Sequência de Carboidratos/genética , Cromatografia Líquida de Alta Pressão , Dissacarídeos/química , Glucosamina/química , Heparina Liase/química , Espectroscopia de Ressonância Magnética , Oligossacarídeos/genética , Polissacarídeo-Liase/química , Trissacarídeos/química
3.
J Gen Virol ; 101(1): 5-20, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31702540

RESUMO

To gain insights into the role of the head-stalk linker region in the fusion triggering, we constructed mutants by deleting or substituting the linker region (115-NGAANNSG-122) of Newcastle disease virus (NDV) haemagglutinin-neuraminidase (HN) with the corresponding sequences of other paramyxoviruses. The results showed that these HN mutants exhibited different levels of fusion-triggering activity, but most of them maintained comparable levels with wide-type HN in both receptor recognition and neuraminidase activity. We tried to figure out reasons for fusion alteration through assessing the expression and the oligomeric state of HN mutants. Moreover, four mutants with significant fusion changes were introduced into the headless HN stem (HN1-123) to intensively investigate the role of the linker region in fusion triggering. Consequently, the stability of HN oligomers and the structural integrity of the 4 helical-bundle of stalk have complicated influences on the alteration of fusion-triggering activities for different mutants. These data suggested that the head-stalk linker could regulate the fusion triggering at both full-length and headless HN levels.

4.
Virus Genes ; 56(1): 37-48, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31768710

RESUMO

Human parainfluenza virus type 3 (HPIV3) causes the majority of childhood viral pneumonia around the world. Fusing the viral and target cell membranes is crucial for its entry into target cells, and the fusion process requires the concerted actions of two viral glycoproteins: hemagglutinin-neuraminidase (HN) and fusion (F) protein. After binding to the cell surface receptor, sialic acids, HN triggers F to undergo large conformational rearrangements to execute the fusion process. Although it has been reported that several domains of F had important impacts on regulating the membrane fusion activity, what role the DI-DII linker (residues 369-374, namely L1 linker) of the HPIV3 F protein plays in the fusion process still remains confused. We have obtained three chimeric mutant proteins (Ch-NDV-L1, Ch-MV-L1, Ch-HPIV1-L1) containing the full length of HPIV3 F protein but their corresponding DI-DII linker derived from the F protein of Newcastle disease virus (NDV), Measles virus (MV), and Human parainfluenza virus type 1 (HPIV1), respectively. One deletion mutant protein (De-L1), whose DI-DII linker was deleted, has been established simultaneously. Then vaccinia virus-T7 RNA polymerase transient expression system and standard plasmid system were utilized to express the mutant F proteins in BHK-21 cells. These four mutants were determined for membrane fusogenic activity, cell surface expression level, and total mutant F protein expression. All of them resulted in a significant reduction in fusogenic activity in all steps of cell-cell membrane fusion process. There was no significant difference in cell surface protein expression level for the mutants compared with wild-type F. The mutant proteins with inability in fusogenic activity were all at the form of precursor protein, F0, which were not hydrolyzed by intracellular protease furin. The results above suggest that the involvement of the DI-DII linker region is necessary for the complete fusion of the membranes.

5.
Virus Res ; 275: 197791, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31628980

RESUMO

Human parainfluenza virus type 3 (HPIV3) fuses the viral envelope with the host cell membrane through the concerted action of the fusion (F) protein and the hemagglutinin-neuraminidase (HN). Upon HN binding to sialic acid (SA), the F protein in a metastable prefusion form is activated to undergo a series of structural rearrangements into a stable postfusion form to actuate the fusion between membranes. Various domains of F protein of some other paramyxoviruses, including HPIV3, have been reported to be differently functional. However, it is not yet clear what roles HRB linker plays. To clarify the roles that HRB linker might play in the F-mediated membrane fusion process, here we examined the effects of mutations introduced into the HRB linker of HPIV3 F protein. Six Single amino acid mutants, three chimeric mutants, and one deletion mutant were obtained and analyzed for membrane fusion activity and cell surface expression. The results showed that the membrane fusion activity of mutants changed to varying degrees in comparison with wild-type (wt) F, and some mutants even forfeited fusogenicity absolutely. It is indicated that the HRB linker domain plays an important role in the F-mediated membrane fusion process.

6.
J Gen Virol ; 100(6): 958-967, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31140969

RESUMO

Newcastle disease (ND), which is caused by Newcastle disease virus (NDV), is a highly contagious disease in chickens and is a great threat to the poultry industry. Fusion of the viral and target cell membranes is a prerequisite for NDV's entry into host cells. This process is directly mediated by the fusion (F) protein. Although several domains of F are known to regulate membrane fusion activity, the roles of the DI-DII linker (residues 376-381) of the NDV F protein in membrane fusion still remain unclear. To investigate the roles of this linker in NDV F-induced cell-cell fusion, mutations were engineered into this linker by site-directed mutagenesis. These mutants were analysed with respect to cell surface expression and membrane fusion activity. Each of the mutated F proteins in this linker was expressed at the cell surface at a similar level to wild-type (WT) F. However, most of them resulted in significant alterations in fusion activity. In particular, the mutants G377S, A378D, L379A and T380P were able to independently mediate cell fusion in the absence of HN protein in BHK-21 cells. Taken together, the results indicated that the DI-DII linker region has an important effect on the fusion activity of NDV F and mutants in this region could alter the requirement for HN for the promotion of membrane fusion.


Assuntos
Hemaglutininas/genética , Proteínas de Fusão de Membrana/genética , Mutação/genética , Neuraminidase/genética , Vírus da Doença de Newcastle/genética , Animais , Fusão Celular/métodos , Linhagem Celular , Membrana Celular/genética , Chlorocebus aethiops , Cricetinae , Doença de Newcastle/virologia , Células Vero
7.
Biosci Trends ; 13(3): 225-233, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31142702

RESUMO

Newcastle disease virus (NDV), an avian paramyxovirus, causes Newcastle disease (ND) which is a highly contagious and fatal viral disease affecting poultry and most species of birds. The fusion (F) protein of NDV mediates membrane fusion, which is essential to the processes of viral entry, replication, and dissemination. Although several domains of NDV F are known to have important effects on regulating the membrane fusion activity, the role of the region around domain III (DIII) and domain I (DI) still remains ill-defined. Site-directed mutagenesis was utilized to change the conserved amino acids at 269, 274, 277, 286, 287, 290, 295, and 297 to alanine in order to investigate the effects of these conserved amino acids around the DIII and DI linker region of the NDV F protein on fusion activity. It was found that five of these substitutions almost abolished fusion activity except for mutants I269A, Q286A, and N297A, which showed 57.1%, 161.1%, and 97.7% of the wt F level, respectively. Four (I274A, D277A, V287A, and P290A) of these five mutants likely result in interfering with folding or transporting of the molecule since these proteins were minimally expressed at the cell surface, formed aggregates, or not proteolytically cleaved. However, mutant L295A almost abolished fusion activity even with a similar level of cell surface expression. These data indicated that conserved amino acids around the DIII-DI linker region are critical for the folding of the F protein and have an important influence on fusion activity.


Assuntos
Vírus da Doença de Newcastle/metabolismo , Proteínas Virais de Fusão/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Animais , Western Blotting , Cricetinae , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Dobramento de Proteína
8.
J Proteomics ; 195: 11-22, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30630120

RESUMO

Oxidized low-density lipoprotein (ox-LDL) can impair endothelial function and lead to the atherosclerosis development. Protein S-nitrosylation is sensitive to cellular redox state and acts as a crucial regulator and executor of nitric oxide (NO) signaling pathways. Aberrant S-nitrosylation contributes to the pathogenesis of cardiovascular and cerebrovascular diseases. However, the effect of ox-LDL on S-nitrosylation and its significance for endothelial dysfunction have not been studied at proteome level. Herein, the combined quantitative analysis of proteome and S-nitrosoproteome was performed using an integrated biotin switch and iTRAQ labeling approach in EA.hy926 cell line derived from human umbilical vein endothelial cell (HUVEC) treated with ox-LDL. A total of 2204 S-nitrosylated (SNO) peptides of 1318 SNO-proteins were quantified. Notably, 352 SNO-peptides of 262 SNO-proteins were significantly regulated after excluding S-nitrosylation changes caused by protein expression alterations. Many of them belonged to mRNA splicing, ribosomal structure and translational regulatory proteins, covering the entire translation process. The results indicated that S-nitrosylation of the splicing and translational machinery in vascular endothelial cells was susceptible to ox-LDL. Abnormal protein S-nitrosylation may be one pivotal mechanism underlying endothelial dysfunction induced by ox-LDL. This study potentially enriches the present understanding of pro-atherogenic effect of ox-LDL from the perspective of S-nitrosylation. SIGNIFICANCE: The role of ox-LDL in endothelial dysfunction and atherosclerosis development has been recognized from the aspect of impaired NO production. However, its effect on S-nitrosylation, which is directly related to NO signaling pathway, still remains largely unexplored. Our work initially provided a systematic characterization of S-nitrosoproteome in ox-LDL-treated endothelial cells after ruling out the changes of S-nitrosylation modification caused by protein expression alone. MS-based approach coupled with iTRAQ technique indicated 262 SNO-proteins were significantly regulated. Functional enrichment and interaction network analysis revealed that proteins involved in mRNA splicing and translational machinery were susceptible to abnormal S-nitrosylation under ox-LDL treatment. This achievement suggested one potential mechanism underlying endothelial dysfunction induced by ox-LDL from the perspective of S-nitrosoproteome.

9.
Carbohydr Polym ; 203: 87-94, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30318238

RESUMO

Heparin is a significant anticoagulant that has been used in clinic over decades. Although numerous efforts have been made to characterize the molecular structure of heparin and its derivatives for safety of the medicine, technical barriers still exist because of their structural complexity. In this study, we have established a method capable to evaluate both the epimerization and composition of heparin and dalteparin by a UHPLC-HILIC/WAX-MS/MS approach. Ten major disaccharide building blocks retaining the epimerization configuration of parental heparin chains were generated and well separated, 9 of which were unambiguously identified. Isomer identifications were achieved through high-resolution tandem mass spectrometry analysis with reference to elaborately prepared standards. The method was successfully applied for the sameness study of generic dalteparins in combination with an isotopic labelling procedure. We believe this robust method maybe adapted to quality control in pharmaceutical production of heparin and LMWHs.

10.
Carbohydr Polym ; 197: 83-91, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30007661

RESUMO

Low molecular weight heparins (LMWHs) are important anticoagulant drugs. Nitrous acid degradation is a major approach to produce LMWHs, such as dalteparin. Due to the foreseeable shortage of porcine intestinal mucosa heparin and other potential risks, expansion of other animal tissues for heparin preparation is necessary. Heparins from different tissues differ in structure and bioactivity potency, and these variations may be carried over to the LMWH products. Sophisticated analytical techniques have been applied to compare various versions of dalteparins produced from porcine intestinal, bovine lung and ovine intestinal heparins to elucidate the effects of different animal tissues starting materials and processing conditions on the properties of final dalteparin products. With adjusted depolymerization conditions, versions of dalteparins that qualify under the European Pharmacopeia (EP) specifications were manufactured using non-porcine heparins. Dissimilarities among the three interspecies animal tissue heparin-derived dalteparins regarding fine structures are also disclosed, and their origins are discussed.


Assuntos
Heparina/síntese química , Heparina/metabolismo , Ácido Nitroso/metabolismo , Animais , Configuração de Carboidratos , Bovinos , Heparina/química , Intestinos/química , Pulmão/química , Peso Molecular , Ácido Nitroso/química , Ovinos
11.
Virus Genes ; 54(3): 333-342, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29516315

RESUMO

Human parainfluenza virus type 3 (hPIV3) is an important respiratory pathogen that causes the majority of viral pneumonia of infants and young children. hPIV3 can infect host cells through the synergistic action of hemagglutinin-neuraminidase (HN) protein and the homotypic fusion (F) protein on the viral surface. HN protein plays a variety of roles during the virus invasion process, such as promoting viral particles to bind to receptors, cleaving sialic acid, and activating the F protein. Crystal structure research shows that HN tetramer adopted a "heads-down" conformation, at least two heads dimmer on flank of the four-helix bundle stalk, which forms a symmetrical interaction interface. The stalk region determines interactions and activation of F protein in specificity, and the heads in down position statically shield these residues. In order to make further research on the function of these amino acids at the hPIV3 HN stalk/head interface, fifteen mutations (8 sites from stalk and 7 sites from head) were engineered into this interface by site-directed mutagenesis in this study. Alanine substitution in this region of hPIV3 HN had various effects on cell fusion promotion, receptor binding, and neuraminidase activity. Besides, L151A also affected surface protein expression efficiency. Moreover, I112A, D120A, and R122A mutations of the stalk region that were masked by global head in down position had influence on the interaction between F and HN proteins.


Assuntos
Aminoácidos/fisiologia , Proteína HN/química , Proteína HN/fisiologia , Vírus da Parainfluenza 3 Humana/química , Vírus da Parainfluenza 3 Humana/fisiologia , Internalização do Vírus , Alanina/química , Linhagem Celular , Membrana Celular/metabolismo , Células Gigantes/virologia , Proteína HN/genética , Hemadsorção , Humanos , Fusão de Membrana/fisiologia , Mutagênese Sítio-Dirigida , Neuraminidase/metabolismo , Vírus da Parainfluenza 3 Humana/genética , Conformação Proteica , Receptores Virais/metabolismo , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/fisiologia
12.
Carbohydr Polym ; 183: 81-90, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29352895

RESUMO

Low molecular weight heparins (LMWHs) are widely used anticoagulant drugs. The composition and sequence of LMWH oligosaccharides determine their safety and efficacy. The short oligosaccharide pool in LMWHs undergoes more depolymerization reactions than the longer chains and is the most sensitive indicator of the manufacturing process. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) has been demonstrated as a powerful tool to sequence synthetic heparin oligosaccharide but never been applied to analyze complicated mixture like LMWHs. We established an offline strong anion exchange (SAX)-high performance liquid chromatography (HPLC) and ESI-MS/MS approach to sequence the short oligosaccharides of dalteparin sodium. With the help of in-house developed MS/MS interpretation software, the sequences of 18 representative species ranging from tetrasaccharide to octasaccharide were obtained. Interestingly, we found a novel 2,3-disulfated hexauronic acid structure and reconfirmed it by complementary heparinase digestion and LC-MS/MS analysis. This approach provides straightforward and in-depth insight to the structure of LMWHs and the reaction mechanism of heparin depolymerization.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Dalteparina/química , Oligossacarídeos/química , Análise de Sequência/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos
13.
Carbohydr Res ; 452: 54-63, 2017 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-29065343

RESUMO

Glycosaminoglycans (GAGs) are a family of linear and anionic polysaccharides that play essential roles in many biological and physiological processes. Interactions between GAGs and proteins regulate function in many proteins and are related to many human diseases and disorders. The structural motifs and mechanisms for interactions between GAGs and proteins are not fully understood. Specific bindings, including minor but unique sequences sporadically distributed along the GAG chains or variably sulfated domains interspersed by undersulfated regions, may be specifically recognized by defined domains of a variety of proteins. Understanding the molecular basis of these interactions will provide a template for developing novel glycotherapeutic agents. The present article reviews recent methodologies and progress on the characterization of structural motifs in both GAGs and proteins involved in GAG-protein interactions. The analytical approaches are categorized into three groups: affinity-based methods; molecular docking, nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography; and mass spectrometry (MS) techniques. The advantages and limitations of each category of methods are discussed and are based on examples of using these techniques to investigate binding between GAGs and proteins.


Assuntos
Glicosaminoglicanos/química , Proteínas/química , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Ligação Proteica
14.
Int J Nanomedicine ; 12: 4443-4454, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28670118

RESUMO

Colla corii asini (CCA) is a protein-based traditional Chinese medicine made from donkey skins. Because it has the ability to nourish blood, its demand is increasing rapidly. The shortage of donkey skins increases the risk of the adulteration of CCA products with other animal skins. To ensure the drug efficacy and safety of CCA products, a proteomics technique was applied to reveal proteins in the skins of donkey, horse, cattle, and pig. Species-specific peptides for each animal species were predicted using bioinformatics, and their presence in the skins and gelatin samples was examined by nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS). One unique marker peptide for each animal species was selected to develop an LC-MS/MS multiple reaction monitoring method. The capability of this method to identify donkey, horse, cattle, and pig materials was demonstrated by analyzing in-house-made donkey gelatins containing different amounts of other animal skins and commercial CCA products. The adulteration of non-donkey species could be sensitively detected at a low level of 0.5%. Hybrid animals, such as mules and hinnies, were also differentiated from donkeys. We provide a practical tool for the quality control of CCA products. The strategy can also be used to study other important traditional Chinese medicines which contain animal proteins.


Assuntos
Cromatografia Líquida/métodos , Colágeno/química , Proteínas/análise , Pele/química , Espectrometria de Massas em Tandem/métodos , Animais , Biomarcadores/análise , Bovinos , Cromatografia Líquida/instrumentação , Colágeno/análise , Equidae , Gelatina/química , Cavalos , Medicina Tradicional Chinesa , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Proteômica/instrumentação , Proteômica/métodos , Especificidade da Espécie , Suínos , Espectrometria de Massas em Tandem/instrumentação
15.
Mol Cell Proteomics ; 16(7): 1233-1243, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28450420

RESUMO

O-GlcNAcylation of carbohydrate-responsive element-binding protein (ChREBP) is believed as an important modulator of ChREBP activities, however little direct evidence of O-GlcNAcylation on ChREBP and no exact O-GlcNAcylation sites have been reported so far. Here, we validate O-GlcNAcylation on ChREBP in cell-free coupled transcription/translation system and in cells by chemoenzymatic and metabolic labeling, respectively. Moreover, for the first time, we identify O-GlcNAcylation on Ser614 in the C-terminus of ChREBP by mass spectrometry and validate two important sites, Thr517 and Ser839 for O-GlcNAcylation and their function via molecular and chemical biological method. Under high glucose conditions, Ser514 phosphorylation enhances ChREBP O-GlcNAcylation, maintaining the transcriptional activity of ChREBP; Ser839 O-GlcNAcylation is essential for Mlx-heterodimerization and DNA-binding activity enhancement, consequently inducing transcriptional activity. Ser839 O-GlcNAcylation is also crucial for ChREBP nuclear export partially by strengthening interactions with CRM1 and 14-3-3. This work is a detailed study of ChREBP O-GlcNAcylation and highlights the biological consequences of the site-specific O-GlcNAcylation dynamics of ChREBP.


Assuntos
Hepatócitos/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Serina/metabolismo , Treonina/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteínas 14-3-3/metabolismo , Transporte Ativo do Núcleo Celular , Acilação , Animais , Sítios de Ligação , Linhagem Celular , Sistema Livre de Células , Glucose/metabolismo , Hepatócitos/citologia , Carioferinas/metabolismo , Espectrometria de Massas , Camundongos , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Receptores Citoplasmáticos e Nucleares/metabolismo
16.
J Am Chem Soc ; 139(14): 5249-5256, 2017 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-28340300

RESUMO

The sulfation at the 3-OH position of glucosamine is an important modification in forming structural domains for heparan sulfate to enable its biological functions. Seven 3-O-sulfotransferase isoforms in the human genome are involved in the biosynthesis of 3-O-sulfated heparan sulfate. As a rare modification present in heparan sulfate, the availability of 3-O-sulfated oligosaccharides is very limited. Here, we report the use of a chemoenzymatic synthetic approach to synthesize six 3-O-sulfated oligosaccharides, including three hexasaccharides and three octasaccharides. The synthesis was achieved by rearranging the enzymatic modification sequence to accommodate the substrate specificity of 3-O-sulfotransferase 3. We studied the impact of 3-O-sulfation on the conformation of the pyranose ring of 2-O-sulfated iduronic acid using NMR, and on the correlation between ring conformation and anticoagulant activity. We identified a novel octasaccharide that interacts with antithrombin and displays anti factor Xa activity. Interestingly, the octasaccharide displays a faster clearance rate than fondaparinux, an FDA-approved pentasaccharide drug, in a rat model, making this octasaccharide a potential short-acting anticoagulant drug candidate that could reduce bleeding risk. Having access to a set of critically important 3-O-sulfated oligosaccharides offers the potential to develop new heparan sulfate-based therapeutics.

17.
Anal Chim Acta ; 961: 91-99, 2017 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-28224913

RESUMO

Heparin is a structurally complex, polysaccharide anticoagulant derived from livestock, primarily porcine intestinal tissues. Low molecular weight (LMW) heparins are derived through the controlled partial depolymerization of heparin. Increased manufacturing and regulatory concerns have provided the motivation for the development of more sophisticated analytical methods for determining both their structure and pedigree. A strategy, for the comprehensive comparison of parent heparins and their LMW heparin daughters, is described that relies on the analysis of monosaccharide composition, disaccharide composition, and oligosaccharide composition. Liquid chromatography-mass spectrometry is rapid, robust, and amenable to automated processing and interpretation of both top-down and bottom-up analyses. Nuclear magnetic resonance spectroscopy provides complementary top-down information on the chirality of the uronic acid residues and glucosamine substitution. Principal component analysis (PCA) was applied to the normalized abundance of oligosaccharides, calculated in the bottom-up analysis, to show parent and daughter correlation in oligosaccharide composition. Using these approaches, six pairs of parent heparins and their daughter generic enoxaparins from two different manufacturers were comprehensively analyzed. Enoxaparin is the most widely used LMW heparin and is prepared through controlled chemical ß-eliminative cleavage of porcine intestinal heparin. Lovenox®, the innovator version of enoxaparin marketed in the US, was analyzed as a reference for the daughter LMW heparins. The results, show similarities between LMW heparins from two different manufacturers with Lovenox®, excellent lot-to-lot consistency of products from each manufacturer, and detects a correlation between each parent heparin and daughter LMW heparin.


Assuntos
Heparina de Baixo Peso Molecular/análise , Heparina/análise , Animais , Cromatografia Líquida , Suínos , Espectrometria de Massas em Tandem
18.
Clin Appl Thromb Hemost ; 23(6): 542-553, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28056526

RESUMO

Heparin and its low-molecular-weight heparin (LMWH) derivatives are widely used clinical anticoagulants. These drugs are critical for the practice of medicine in applications including kidney dialysis, cardiopulmonary bypass, and in the management of venous thromboembolism. Currently, these drugs are derived from livestock, primarily porcine intestine. The worldwide dependence on a single animal species has made the supply chain for this critical drug quite fragile, leading to the search for other sources of these drugs, including bovine tissues such as bovine intestine or lung. A number of laboratories are currently examining the similarities and differences between heparins prepared from porcine and bovine tissues. The current study is designed to compare LMWH prepared from bovine heparins through chemical ß-elimination, a process currently used to prepare the LMWH, enoxaparin, from porcine heparin. Using top-down, bottom-up, compositional analysis and bioassays, LMWHs, derived from bovine lung and intestine, are shown to closely resemble enoxaparin.


Assuntos
Enoxaparina/normas , Heparina de Baixo Peso Molecular/normas , Animais , Anticoagulantes/uso terapêutico , Bovinos , Técnicas de Laboratório Clínico , Heparina de Baixo Peso Molecular/uso terapêutico , Intestinos/química , Pulmão/química , Suínos
19.
J Chromatogr A ; 1479: 121-128, 2017 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-27923477

RESUMO

Low molecular weight heparins (LMWHs) are important anticoagulant drugs that are prepared through depolymerization of unfractionated heparin. Based on the types of processing reactions and the structures of the products, LMWHs can be divided into different classifications. Enoxaparin is prepared by benzyl esterification and alkaline depolymerization, while dalteparin and nadroparin are prepared through nitrous acid depolymerization followed by borohydride reduction. Compositional analysis of their basic building blocks is an effective way to provide structural information on heparin and LMWHs. However, most current compositional analysis methods have been limited to heparin and enoxaparin. A sensitive and comprehensive approach is needed for detailed investigation of the structure of LMWHs prepared through nitrous acid depolymerization, especially their characteristic saturated non-reducing end (NRE) and 2,5-anhydro-d-mannitol reducing end (RE). A maltose modified hydrophilic interaction column offers improved separation of complicated mixtures of acidic disaccharides and oligosaccharides. A total of 36 basic building blocks were unambiguously identified by high-resolution tandem mass spectrometry (MS). Multiple reaction monitoring (MRM) MS/MS quantification was developed and validated in the analysis of dalteparin and nadroparin samples. Each group of building blocks revealed different aspects of the properties of LMWHs, such as functional motifs required for anticoagulant activity, the structure of heparin starting materials, cleavage sites in the depolymerization reaction, and undesired structural modifications resulting from side reactions.


Assuntos
Heparina de Baixo Peso Molecular/química , Heparina/química , Ácido Nitroso/química , Cromatografia Líquida , Heparina/metabolismo , Heparina Liase/metabolismo , Heparina de Baixo Peso Molecular/isolamento & purificação , Interações Hidrofóbicas e Hidrofílicas , Nadroparina/análise , Espectrometria de Massas em Tandem
20.
Anal Bioanal Chem ; 409(2): 411-420, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27325464

RESUMO

Most hyphenated analytical approaches that rely on liquid chromatography-MS require relatively long separation times, produce incomplete resolution of oligosaccharide mixtures, use eluents that are incompatible with electrospray ionization, or require oligosaccharide derivatization. Here we demonstrate the analysis of heparin oligosaccharides, including disaccharides, ultralow molecular weight heparin, and a low molecular weight heparin, using a novel electrokinetic pump-based CE-MS coupling eletrospray ion source. Reverse polarity CE separation and negative-mode electrospray ionization were optimized using a volatile methanolic ammonium acetate electrolyte and sheath fluid. The online CE hyphenated negative-ion electrospray ionization MS on an LTQ Orbitrap mass spectrometer was useful in disaccharide compositional analysis and bottom-up and top-down analysis of low molecular weight heparin. The application of this CE-MS method to ultralow molecular heparin suggests that a charge state distribution and the low level of sulfate group loss that is achieved make this method useful for online tandem MS analysis of heparins. Graphical abstract Most hyphenated analytical approaches that rely on liquid chromatography-MS require relatively long separation times, produce incomplete resolution of oligosaccharide mixtures, use eluents that are incompatible with electrospray ionization, or require oligosaccharide derivatization. Here we demonstrate the analysis of heparin oligosaccharides, including disaccharides, ultralow molecular weight heparin, and a low molecular weight heparin, using a novel electrokinetic pump-based CE-MS coupling eletrospray ion source. Reverse polarity CE separation and negative-mode electrospray ionization were optimized using a volatile methanolic ammonium acetate electrolyte and sheath fluid. The online CE hyphenated negative-ion electrospray ionization MS on an LTQ Orbitrap mass spectrometer was useful in disaccharide compositional analysis and bottom-up and top-down analysis of low molecular weight heparin. The application of this CE-MS method to ultralow molecular heparin suggests that a charge state distribution and the low level of sulfate group loss that is achieved make this method useful for online tandem MS analysis of heparins.


Assuntos
Técnicas de Química Analítica/métodos , Eletroforese Capilar , Heparina/química , Oligossacarídeos/química , Espectrometria de Massas por Ionização por Electrospray , Técnicas de Química Analítica/instrumentação , Heparina de Baixo Peso Molecular/química
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