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1.
Nat Commun ; 11(1): 2688, 2020 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-32461612

RESUMO

Severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) are zoonotic pathogens with high fatality rates and pandemic potential. Vaccine development focuses on the principal target of the neutralizing humoral immune response, the spike (S) glycoprotein. Coronavirus S proteins are extensively glycosylated, encoding around 66-87 N-linked glycosylation sites per trimeric spike. Here, we reveal a specific area of high glycan density on MERS S that results in the formation of oligomannose-type glycan clusters, which were absent on SARS and HKU1 CoVs. We provide a comparison of the global glycan density of coronavirus spikes with other viral proteins including HIV-1 envelope, Lassa virus glycoprotein complex, and influenza hemagglutinin, where glycosylation plays a known role in shielding immunogenic epitopes. Overall, our data reveal how organisation of glycosylation across class I viral fusion proteins influence not only individual glycan compositions but also the immunological pressure across the protein surface.


Assuntos
Glicoproteínas/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio , Polissacarídeos , Glicoproteína da Espícula de Coronavírus/imunologia , Proteínas Virais de Fusão/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Microscopia Crioeletrônica , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Glicoproteínas/química , Glicoproteínas/ultraestrutura , Glicosilação , Células HEK293 , HIV-1/imunologia , HIV-1/metabolismo , Humanos , Evasão da Resposta Imune/fisiologia , Vírus Lassa/imunologia , Vírus Lassa/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Orthomyxoviridae/imunologia , Orthomyxoviridae/metabolismo , Polissacarídeos/química , Polissacarídeos/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/ultraestrutura , Proteínas Virais/química , Proteínas Virais/imunologia , Proteínas Virais/ultraestrutura
2.
J Phys Chem Lett ; 11(12): 4785-4790, 2020 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-32463239

RESUMO

The severe acute respiratory syndrome coronavirus (SARS-CoV-2) pandemic is setting the global health crisis of our time, causing a devastating societal and economic burden. An idiosyncratic trait of coronaviruses is the presence of spike glycoproteins on the viral envelope, which mediate the virus binding to specific host receptor, enabling its entry into the human cells. In spite of the high sequence identity of SARS-CoV-2 with its closely related SARS-CoV emerged in 2002, the atomic-level determinants underlining the molecular recognition of SARS-CoV-2 to the angiotensin-converting enzyme 2 (ACE2) receptor and, thus, the rapid virus spread into human body, remain unresolved. Here, multi-microsecond-long molecular dynamics simulations enabled us to unprecedentedly dissect the key molecular traits liable of the higher affinity/specificity of SARS-CoV-2 toward ACE2 as compared to SARS-CoV. This supplies a minute per-residue contact map underlining its stunningly high infectivity. Harnessing this knowledge is pivotal for urgently developing effective medical countermeasures to face the ongoing global health crisis.


Assuntos
Betacoronavirus/metabolismo , Glicoproteínas/metabolismo , Simulação de Dinâmica Molecular , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Glicoproteínas/química , Humanos , Ligação de Hidrogênio , Pandemias , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Teoria Quântica , Vírus da SARS/metabolismo , Proteínas Virais/química , Ligação Viral
3.
PLoS One ; 15(5): e0233492, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469948

RESUMO

Glycosylation can affect various protein properties such as stability, biological activity, and immunogenicity. To produce human therapeutic proteins, a host that can produce glycoproteins with correct glycan structures is required. Microbial expression systems offer economical, rapid and serum-free production and are more amenable to genetic manipulation. In this study, we developed a protocol for CRISPR/Cas9 multiple gene knockouts and knockins in Kluyveromyces marxianus, a probiotic yeast with a rapid growth rate. As hyper-mannosylation is a common problem in yeast, we first knocked out the α-1,3-mannosyltransferase (ALG3) and α-1,6-mannosyltransferase (OCH1) genes to reduce mannosylation. We also knocked out the subunit of the telomeric Ku domain (KU70) to increase the homologous recombination efficiency of K. marxianus. In addition, we knocked in the MdsI (α-1,2-mannosidase) gene to reduce mannosylation and the GnTI (ß-1,2-N-acetylglucosaminyltransferase I) and GnTII genes to produce human N-glycan structures. We finally obtained two strains that can produce low amounts of the core N-glycan Man3GlcNAc2 and the human complex N-glycan Man3GlcNAc4, where Man is mannose and GlcNAc is N-acetylglucosamine. This study lays a cornerstone of glycosylation engineering in K. marxianus toward producing human glycoproteins.


Assuntos
Kluyveromyces/genética , Kluyveromyces/metabolismo , Engenharia Metabólica/métodos , Polissacarídeos/biossíntese , Polissacarídeos/química , Biotecnologia , Sistemas CRISPR-Cas , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Genes Fúngicos , Glicoproteínas/biossíntese , Glicoproteínas/química , Glicoproteínas/genética , Glicosilação , Humanos , Manosidases/genética , Manosidases/metabolismo , Manosiltransferases/antagonistas & inibidores , Manosiltransferases/genética , Manosiltransferases/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
4.
J Chromatogr A ; 1620: 461001, 2020 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-32151415

RESUMO

Mass spectrum (MS) is one of the most commonly used tools for qualitative and quantitative analysis of glycans. However, due to the complexity of biological samples and the low ionization efficiency of glycans, these need to be purified and derivatized prior to MS analysis. Existing purification strategies require a combination of multiple methods and are cumbersome to operate. Here, we propose a new method for the purification of glycoprotein N/O-glycans and their derivatives using a hand-packed absorbent cotton hydrophilic interaction chromatography column (HILIC). The method's reliability and applicability were verified by purifying N/O-glycans and the derivatives of standard glycoproteins, such as chicken albumin and porcine stomach mucin. Stable isotope labelling was used to compare the glycans' recovery following different purification methods. Absorbent cotton HILIC was also successfully applied for the analysis of human serum and fetal bovine serum glycoprotein N-glycans. Finally, testing revealed high binding capacity (9 mg/g-1 maltohexaose/absorbent cotton) and good recovery (average recovery was 91.7%) of glycans. Compared with traditional procedures, the proposed purification method offers considerable advantages, such as simplicity, high efficiency, economy, universality, and broad applicability for the pretreatment of glycans and their derivatives in biological samples prior to MS analysis.


Assuntos
Cromatografia/métodos , Polissacarídeos/isolamento & purificação , Animais , Galinhas , Fibra de Algodão , Glicoproteínas/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Marcação por Isótopo , Espectrometria de Massas , Mucinas/química , Polissacarídeos/sangue , Polissacarídeos/química , Suínos
6.
J Biomed Sci ; 27(1): 33, 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-32059697

RESUMO

Vaccination is the most effective measure at preventing influenza virus infections. However, current seasonal influenza vaccines are only protective against closely matched circulating strains. Even with extensive monitoring and annual reformulation our efforts remain one step behind the rapidly evolving virus, often resulting in mismatches and low vaccine effectiveness. Fortunately, many next-generation influenza vaccines are currently in development, utilizing an array of innovative techniques to shorten production time and increase the breadth of protection. This review summarizes the production methods of current vaccines, recent advances that have been made in influenza vaccine research, and highlights potential challenges that are yet to be overcome. Special emphasis is put on the potential role of glycoengineering in influenza vaccine development, and the advantages of removing the glycan shield on influenza surface antigens to increase vaccine immunogenicity. The potential for future development of these novel influenza vaccine candidates is discussed from an industry perspective.


Assuntos
Glicoproteínas/imunologia , Imunogenicidade da Vacina , Vacinas contra Influenza/imunologia , Engenharia de Proteínas , Proteínas Virais/imunologia , Glicoproteínas/química , Glicoproteínas/farmacologia , Glicosilação , Humanos , Vacinas contra Influenza/análise , Vacinas contra Influenza/química , Vacinas contra Influenza/farmacologia , Proteínas Virais/química , Proteínas Virais/farmacologia
7.
J Dairy Sci ; 103(4): 3017-3024, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32089302

RESUMO

Xinong Saanen goat milk is a major source of milk in the Chinese dairy industry. Milk fat globule membrane (MFGM) proteomes of goat colostrum and mature milk were analyzed and compared using proteomic technology. A total of 543 and 585 proteins were identified in goat colostrum and mature milk, respectively. Functional category analyses revealed that most of the MFGM proteins in both colostrum and mature milk were related to phosphoprotein and acetylation. The biological process of translation, cellular component of extracellular exosome, and molecular function of poly(A) RNA binding were the main gene ontology annotations of both colostrum and mature milk. Pathways associated with disease and genetic information processing involved large number of proteins in colostrum and mature milk, and more metabolism-related pathways were observed in mature milk. Protein-protein interaction network analyses showed that ribosome was abundant in both colostrum and mature milk. Colostrum showed more functions associated with protein processing in the endoplasmic reticulum, whereas mature milk had more oxidative phosphorylation functions. The results could provide further understanding of the unique biological properties of MFGM proteins of goat colostrum and mature milk.


Assuntos
Colostro/química , Glicolipídeos/química , Glicoproteínas/química , Cabras , Leite/química , Proteoma , Animais , Feminino , Ontologia Genética , Cabras/metabolismo , Membranas , Proteínas do Leite/análise , Gravidez , Espectrometria de Massas em Tandem
8.
Immunity ; 52(2): 388-403.e12, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32023489

RESUMO

Structural principles underlying the composition of protective antiviral monoclonal antibody (mAb) cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic mAb cocktail against Ebola virus. We systematically analyzed the antibody repertoire in human survivors and identified a pair of potently neutralizing mAbs that cooperatively bound to the ebolavirus glycoprotein (GP). High-resolution structures revealed that in a two-antibody cocktail, molecular mimicry was a major feature of mAb-GP interactions. Broadly neutralizing mAb rEBOV-520 targeted a conserved epitope on the GP base region. mAb rEBOV-548 bound to a glycan cap epitope, possessed neutralizing and Fc-mediated effector function activities, and potentiated neutralization by rEBOV-520. Remodeling of the glycan cap structures by the cocktail enabled enhanced GP binding and virus neutralization. The cocktail demonstrated resistance to virus escape and protected non-human primates (NHPs) against Ebola virus disease. These data illuminate structural principles of antibody cooperativity with implications for development of antiviral immunotherapeutics.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Glicoproteínas/imunologia , Doença pelo Vírus Ebola/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Linhagem Celular , Modelos Animais de Doenças , Quimioterapia Combinada , Epitopos , Feminino , Glicoproteínas/química , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mimetismo Molecular , Conformação Proteica
9.
PLoS One ; 15(2): e0228265, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32012183

RESUMO

Hypertension is considered as one of the most common diseases that affect human beings (both male and female) due to its high prevalence and also extending widely to both industrialize and developing countries. Angiotensin-converting enzyme (ACE) has a significant role in the regulation of blood pressure and ACE inhibition with inhibitory peptides is considered as a major target to prevent hypertension. In the current study, a blood pressure regulating honey protein (MRJP1) was examined to identify the ACE inhibitory peptides. The 3D structure of MRJP1 was predicted by utilizing the threading approach and further optimized by performing molecular dynamics simulation for 30 nanoseconds (ns) to improve the quality factor up to 92.43%. Root mean square deviation and root mean square fluctuations were calculated to evaluate the structural features and observed the fluctuations in the timescale of 30 ns. AHTpin server based on scoring vector machine of regression models, proteolysis and structural characterization approaches were implemented to identify the potential inhibitory peptides. The anti-hypertensive peptides were scrutinized based on the QSAR models of anti-hypertensive activity and the molecular docking analyses were performed to explore the binding affinities and potential interacting residues. The peptide "EALPHVPIFDR" showed the strong binding affinity and higher anti-hypertensive activity along with the global energy of -58.29 and docking score of 9590. The aromatic amino acids especially Tyr was observed as the key residue to design the dietary peptides and drugs like ACE inhibitors.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Simulação por Computador , Glicoproteínas/química , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/farmacologia , Peptidil Dipeptidase A/metabolismo , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/química , Fragmentos de Peptídeos/química , Peptidil Dipeptidase A/química , Domínios Proteicos
10.
Microb Cell Fact ; 19(1): 7, 2020 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-31931833

RESUMO

BACKGROUND: Therapeutic glycoproteins have occupied an extremely important position in the market of biopharmaceuticals. N-Glycosylation of protein drugs facilitates them to maintain optimal conformations and affect their structural stabilities, serum half-lives and biological efficiencies. Thus homogeneous N-glycoproteins with defined N-glycans are essential in their application in clinic therapeutics. However, there still remain several obstacles to acquire homogeneous N-glycans, such as the high production costs induced by the universal utilization of mammalian cell expression systems, the non-humanized N-glycan structures and the N-glycosylation microheterogeneities between batches. RESULTS: In this study, we constructed a Pichia pastoris (Komagataella phaffii) expression system producing truncated N-GlcNAc-modified recombinant proteins through introducing an ENGase isoform (Endo-T) which possesses powerful hydrolytic activities towards high-mannose type N-glycans. The results showed that the location of Endo-T in different subcellular fractions, such as Endoplasmic reticulum (ER), Golgi or cell membrane, affected their hydrolytic efficiencies. When the Endo-T was expressed in Golgi, the secreted IgG1-Fc region was efficiently produced with almost completely truncated N-glycans and the N-GlcNAc modification on the glycosite Asn297 was confirmed via Mass Spectrometry. CONCLUSION: This strategy develops a simple glycoengineered yeast expression system to produce N-GlcNAc modified proteins, which could be further extended to different N-glycan structures. This system would provide a prospective platform for mass production of increasing novel glycoprotein drugs.


Assuntos
Glicoproteínas/biossíntese , Engenharia Metabólica/métodos , Pichia/metabolismo , Polissacarídeos/biossíntese , Produtos Biológicos , Biotecnologia , Glicoproteínas/química , Glicosilação , Pichia/genética , Polissacarídeos/química , Proteínas Recombinantes/biossíntese , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
11.
Food Chem ; 314: 126160, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31958749

RESUMO

Lipolysis products released during digestion exert positive metabolic impacts on the nutrition of newborns. However, the lipolysis behavior of yak milk lipids during digestion remains unknown. In this study, the simulated in vitro infant gastrointestinal digestion of cow, yak and standardized yak milk fat globules the same size as those from cow milk (Cow MF, Yak MF and Yak SMF) were compared. Although Cow MF showed a higher lipolysis rate at the beginning of gastric digestion, Yak MF and Yak SMF exhibited a higher lipolysis level during later gastrointestinal digestion. Higher hydrolysis efficiency of yak milk lipids was due to their lipid properties, including their composition and structure. Furthermore, yak milk lipids released more unsaturated fatty acids than Cow MF throughout digestion. This study highlights the crucial role of lipid characteristics in the efficient digestion of milk lipids and provides new insight for the design of yak milk infant diets.


Assuntos
Digestão , Glicolipídeos/farmacocinética , Glicoproteínas/farmacocinética , Leite/química , Animais , Bovinos , Ácidos Graxos Insaturados/farmacocinética , Feminino , Glicolipídeos/química , Glicoproteínas/química , Humanos , Hidrólise , Lactente , Recém-Nascido , Lipídeos/química , Lipólise
12.
Eur J Med Chem ; 187: 111921, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31835168

RESUMO

The emergence of disease and dearth of effective pharmacological agents on most therapeutic fronts, constitutes a major threat to global public health and man's existence. Consequently, this has created an exigency in the search for new drugs with improved clinical utility or means of potentiating available ones. To this end, accumulating empirical evidence supports molecular target therapy as a plausible egress and, ß-glucuronidase (ßGLU) - a lysosomal acid hydrolase responsible for the catalytic deconjugation of ß-d-glucuronides has emerged as a viable molecular target for several therapeutic applications. The enzyme's activity level in body fluids is also deemed a potential biomarker for the diagnosis of some pathological conditions. Moreover, due to its role in colon carcinogenesis and certain drug-induced dose-limiting toxicities, the development of potent inhibitors of ßGLU in human intestinal microbiota has aroused increased attention over the years. Nevertheless, although our literature survey revealed both natural products and synthetic scaffolds as potential inhibitors of the enzyme, only few of these have found clinical utility, albeit with moderate to poor pharmacokinetic profile. Hence, in this review we present a compendium of exploits in the present millennium directed towards the inhibition of ßGLU. The aim is to proffer a platform on which new scaffolds can be modelled for improved ßGLU inhibitory potency and the development of new therapeutic agents in consequential.


Assuntos
Glucuronidase/antagonistas & inibidores , Glicoproteínas/farmacologia , Relação Dose-Resposta a Droga , Glucuronidase/metabolismo , Glicoproteínas/química , Humanos , Estrutura Molecular , Relação Estrutura-Atividade
13.
Food Chem ; 310: 125949, 2020 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31837532

RESUMO

The effect of interfacial competitive adsorption of glyceryl monostearate (GMS) with proteins and GMS-fat (anhydrous milk fat; coconut oil) interactions on the fat crystallization behavior and stability of whipped-frozen emulsions were investigated. The results indicated GMS retarded the nucleation of emulsified anhydrous milk fat, but accelerated crystal growth. A contrasting outcome was elicited by emulsified coconut oil. Increasing GMS concentration strengthened and weakened the structural networking within anhydrous milk fat and coconut oil emulsions, respectively, which was evidenced by the oscillatory rheology results. Anhydrous milk fat whipped-frozen emulsions were characterized by increased partial coalescence degree with increasing GMS concentration. However, lower partial destabilization index and insignificant effect of GMS was found in coconut oil systems. Confocal laser scanning micrographs revealed that big clumps of fat globules were present at air bubble surfaces in anhydrous milk fat whipped-frozen emulsions, while only some individual fat globules were observed in coconut oil systems.


Assuntos
Glicerídeos/química , Sorvetes , Proteínas/química , Adsorção , Ar , Animais , Óleo de Coco/química , Cristalização , Emulsões/química , Congelamento , Glicolipídeos/química , Glicoproteínas/química , Leite/química , Tamanho da Partícula , Reologia
14.
Chem Biodivers ; 17(1): e1900436, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31705573

RESUMO

A facile method was developed for synthesis of boronic acid-functionalized silica nanocomposites (SiO2 -BA) by 'thiol-ene' click reaction, where silica nanoparticles were synthesized by using tetraethoxysilane (TEOS) and γ-mercaptopropyl trimethoxysilane (γ-MPTS) as precursors. The morphology and structure properties of the resultant SiO2 -BA were characterized by transmission electronic microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), and Brunner-Emmet-Teller measurements (BET). The adsorption behavior of the SiO2 -BA for glycoproteins was evaluated. Under the optimized conditions, the SiO2 -BA exhibited higher adsorption capacity towards glycoproteins (ovalbumin, OVA, 7.64 µmol/g) than non-glycoproteins (bovine serum albumin, BSA, 0.83 µmol/g). In addition, the practicality of the SiO2 -BA was further assessed by selective enrichment of glycoproteins from egg white samples.


Assuntos
Ácidos Borônicos/química , Glicoproteínas/química , Nanocompostos/química , Dióxido de Silício/química , Adsorção , Clara de Ovo/química , Estrutura Molecular , Tamanho da Partícula , Dióxido de Silício/síntese química , Propriedades de Superfície
15.
Protein Expr Purif ; 166: 105503, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31550499

RESUMO

The N-glycosylation process that occurs in the Pichia pastoris protein expression system can have a significant effect on the yield of heterologous glycoproteins secreted from the yeast. The basis of the effect of N-glycosylation on yield, however, has not been elucidated. In order to investigate the effect of N-glycosylation on heterologous protein production, site-directed mutation was performed on five potential N-glycosylation sites of the tetanus toxin fragment C (TetC). Unaltered TetC (wild-TetC) and eight mutants, in which different numbers and locations of N-glycosylation sites were altered, were expressed in P. pastoris GS115. The recombinant target proteins presented different levels of N-glycosylation. The wild Tet-C and 4 mutations sites of putative N-glycosylation (4Gly mutant: N280Q) had the highest level of secreted protein, while 1 mutation of putative N-glycosylation sites (1Gly mutant: N39/64/85/205Q) had the highest level of intracellular, non-secreted heterologous protein. Reducing the number of native N-glycosylation sites decreased the level of glycosylation, as well as the level of secretion. Introduction of a N-glycosylation site at position 320, however, also reduced the level of expression and secretion of recombinant protein. These results indicate that the number and location of N-glycosylation sites jointly have an effect on the expression and secretion of heterologous glycoproteins in P. pastoris.


Assuntos
Glicoproteínas/genética , Fragmentos de Peptídeos/genética , Pichia/genética , Proteínas Recombinantes/genética , Toxina Tetânica/genética , Sequência de Aminoácidos , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos/genética , Glicoproteínas/química , Glicosilação , Mutagênese Sítio-Dirigida , Mutação , Fragmentos de Peptídeos/química , Pichia/enzimologia , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Toxina Tetânica/química , Transfecção
16.
J Dairy Sci ; 103(3): 2487-2497, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31882218

RESUMO

Lactoferrin (LF) and milk fat globule (MFG) are 2 biologically active components of milk with great economical and nutritional value in the dairy industry. The objectives of this study were to estimate (1) the heritability of mid-infrared (MIR)-predicted LF and MFG size (MFGS) and (2) the genetic correlations between predicted LF and MFGS with milk, fat, and protein yields, fat and protein percentages, and somatic cell score in first-parity Canadian Holstein cattle. A total of 109,029 test-day records from 22,432 cows and 1,572 farms for MIR-predicted LF and 109,212 test-day records from 22,424 cows and 1,559 farms for MIR-predicted MFGS were used in the analyses. Four separate 5-trait random regression test-day models were used. The models included days in milk, herd test date, and a polynomial regression on DIM nested in age-season of calving classes as fixed effects, random polynomial regressions on DIM nested in herd-year of calving, animal additive genetic and permanent environment classes, and a residual effect. Regression curves were modeled using orthogonal Legendre polynomials of order 4 for the fixed age-season of calving effect and of order 5 for the random effects. Moderate overall heritability estimates of 0.34 and 0.46 were estimated for the MIR-predicted LF and MIR-predicted MFGS, respectively. These heritability estimates were similar to the ones estimated for the direct measure of MFGS in a previous study. The genetic correlations between predicted MFGS and fat percentage (0.53) and between predicted LF and protein percentage (0.41) were both moderate and positive. Predicted LF and somatic cell score showed a weaker correlation (0.06) compared with other studies. The moderate genetic correlation between MIR-predicted MFGS and fat percentage and between MIR-predicted LF and protein percentage suggests that MIR predictions of MFGS and LF are not simply a function of the amount of fat and protein percentage, respectively, in the milk (i.e., the prediction equations are not simply predicting fat or protein percentages). Thus, these MIR-predicted values may provide additional information for selecting for fine milk components in Holstein cattle.


Assuntos
Bovinos/genética , Glicolipídeos/metabolismo , Glicoproteínas/metabolismo , Lactação , Lactoferrina/metabolismo , Leite/química , Animais , Canadá , Bovinos/metabolismo , Indústria de Laticínios , Feminino , Glicolipídeos/química , Glicoproteínas/química , Padrões de Herança , Lactação/genética , Lactoferrina/química , Paridade , Fenótipo , Gravidez , Espectrofotometria Infravermelho/veterinária
17.
J Dairy Sci ; 103(1): 179-190, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31733849

RESUMO

Fat separation is a limiting factor for the shelf life of UHT milk. It may be promoted by the proteolysis of fat surface-adsorbed proteins (FSAP) by proteases that remain active after UHT treatment. The aim of this research was to explore the relationship between the proteolysis of FSAP and fat destabilization. In this study, we developed a full-fat UHT milk-based model system and added either the major bacterial protease AprX from Pseudomonas fluorescens or the major native milk protease plasmin at high levels to induce fast destabilization of the milk fat globules. We monitored changes in physical properties and FSAP composition, and structural changes in fat globules, over 24 h. Our results showed that AprX-induced sedimentation as a result of the flocculation of fat globules, and plasmin induced cream to float as a result of the coalescence of fat globules. This study confirmed that AprX and plasmin can both lead to fat destabilization in full-fat UHT milk, and it provides insights in the underlying mechanisms.


Assuntos
Proteínas de Bactérias/metabolismo , Fibrinolisina/análise , Glicolipídeos/química , Glicoproteínas/química , Leite/enzimologia , Peptídeo Hidrolases/metabolismo , Pseudomonas fluorescens/enzimologia , Serina Endopeptidases/metabolismo , Animais , Proteínas de Bactérias/genética , Leite/química , Proteólise , Pseudomonas fluorescens/fisiologia , Serina Endopeptidases/genética
18.
Carbohydr Polym ; 229: 115528, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31826422

RESUMO

Carbohydrate sequences are important for various biological processes. It has recently been estimated to have 100,000-500,000 carbohydrate structures in mammalian glycome. However, the peripheral carbohydrate determinants on N- and O-glycoproteins, glycolipids, polysaccharides and secreted free sugars are limited in numbers. Among these blood-group-related antigens the ABO(H)- and Lewis-types are particularly important. Negative-ion MS/MS has been successfully used in assignment of these epitopes on free reducing sugars but cannot be applied to reduced sugars, e.g. O-glycans typically released from mucins as alditols, or in positive-ion detection of either reducing or reduced oligosaccharides. In the present study, we investigate the fragmentation features of permethylated reducing and reduced sugars under positive-ion conditions of multi-stage MALDI-MS, and propose the concept of epitope ion and epitope spectrum for determination of peripheral blood-group related epitopes on secreted human milk oligosaccharides and N-glycans as reducing sugars and O-glycans as reduced alditols in conjunction with MALDI-MS glycan profiling.


Assuntos
Epitopos/análise , Glicoproteínas/química , Oligossacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sistema ABO de Grupos Sanguíneos/química , Sistema ABO de Grupos Sanguíneos/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Mucosa Gástrica/metabolismo , Humanos , Íons/química , Antígenos do Grupo Sanguíneo de Lewis/química , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Leite Humano/química , Leite Humano/metabolismo , Mucinas/química , Mucinas/metabolismo , Álcoois Açúcares/química , Suínos
19.
Food Chem ; 309: 125671, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31670129

RESUMO

A pure glycoprotein (BGP4-I) was obtained from tartary buckwheat seeds by aqueous extraction followed by DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-100 gel filtration chromatography. The average molecular weight of BGP4-I, as determined by high performance gel permeation chromatography, was 123.43 kDa. The structure of BGP4-I was characterized based on Fourier transform infrared spectroscopy, circular dichroism spectroscopy, and nuclear magnetic resonance spectroscopy, etc. Based on the nano-liquid chromatography-coupled electrospray ionization mass spectrometry analysis of the amino acid sequence of BGP4-I, belongs unequivocally to the glycosyl hydrolase family 1 in the Carbohydrate Active Enzymes database by alignment studies. The specific activity of BGP4-I was 18.44 µmol/min/mg on the substrate p-nitrophenyl-ß-d-glucopyranoside. Furthermore, BGP4-I is unique in its specificity for some substrates. These results suggest that the BGP4-I from tartary buckwheat seeds is a novel specific ß-glucosidase setting the foundation for potential applications in the food industry.


Assuntos
Fagopyrum/metabolismo , Glicoproteínas/química , Proteínas de Plantas/química , Sementes/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Peso Molecular , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/isolamento & purificação , N-Glicosil Hidrolases/metabolismo , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Especificidade por Substrato , Espectrometria de Massas em Tandem
20.
Talanta ; 206: 120178, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514832

RESUMO

A facile and mild approach was carried out to synthesize citrate acid-magnetic ferroferric oxide for glycopeptide analysis. The material was synthesized successfully and applied in glycopeptide identification from human saliva, indicating that this method could be a promising tool for glycopeptidome analysis, which also enlightened the simple fabrication of hydrophilic materials in analytical science.


Assuntos
Ácido Cítrico/química , Glicopeptídeos/análise , Nanopartículas de Magnetita/química , Saliva/química , Cromatografia Líquida/métodos , Glicoproteínas/química , Humanos , Limite de Detecção , Fragmentos de Peptídeos/análise , Proteólise , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Tripsina/química
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