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1.
Biologicals ; 62: 77-84, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31522909

RESUMO

Multiple analytical and preclinical studies were performed to compare the biochemical characteristics, pharmacokinetics (PK), safety and neoantigenicity of a new 5% liquid formulation of Alpha-1 Proteinase Inhibitor (Liquid A1PI, Prolastin®-C Liquid) with the lyophilized version (Lyophilized A1PI, Prolastin®-C). Liquid A1PI and Lyophilized A1PI had similar average mass (~52 kDa), and both forms exhibited glycoform patterns consistent with the known banding pattern of A1PI (dominated by the M6 and M4 bands, including deconvoluted masses). Both Liquid A1PI and Lyophilized A1PI yielded average percent purity values ranging from 96% to 99% and had active content ranging from 53  mg/mL to 59  mg/mL. The PK profile of Liquid A1PI was similar to Lyophilized A1PI. Safety assessments in rabbits showed good tolerability and no test article-related changes in mortality, clinical signs, clinical pathology, body weight, food consumption, or urinalysis parameters. Following immunodepletion of antibodies that recognize Lyophilized A1PI, there were no significant differences in the anti-drug titers among animals immunized with Lyophilized A1PI and Liquid A1PI (p > 0.05), indicating that no antibodies to neoantigens were generated. Liquid A1PI and Lyophilized A1PI have similar profiles with respect to biochemical characteristics, PK, safety and neoantigenicity.


Assuntos
Deficiência de alfa 1-Antitripsina/tratamento farmacológico , alfa 1-Antitripsina , Animais , Anticorpos/sangue , Anticorpos/imunologia , Liofilização , Humanos , Coelhos , alfa 1-Antitripsina/efeitos adversos , alfa 1-Antitripsina/imunologia , alfa 1-Antitripsina/farmacocinética , alfa 1-Antitripsina/farmacologia , Deficiência de alfa 1-Antitripsina/sangue , Deficiência de alfa 1-Antitripsina/imunologia
2.
Biophys J ; 110(8): 1732-1743, 2016 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-27119634

RESUMO

Dynamic water solvation is crucial to protein conformational reorganization and hence to protein structure and functionality. We report here the characterization of water dynamics on the L-asparaginase structural homology isozymes L-asparaginases I (AnsA) and II (AnsB), which are shown via fluorescence spectroscopy and dynamics in combination with molecular dynamics simulation to have distinct catalytic activity. By use of the tryptophan (Trp) analog probe 2,7-diaza-tryptophan ((2,7-aza)Trp), which exhibits unique water-catalyzed proton-transfer properties, AnsA and AnsB are shown to have drastically different local water environments surrounding the single Trp. In AnsA, (2,7-aza)Trp exhibits prominent green N(7)-H emission resulting from water-catalyzed excited-state proton transfer. In stark contrast, the N(7)-H emission is virtually absent in AnsB, which supports a water-accessible and a water-scant environment in the proximity of Trp for AnsA and AnsB, respectively. In addition, careful analysis of the emission spectra and corresponding relaxation dynamics, together with the results of molecular dynamics simulations, led us to propose two structural states associated with the rearrangement of the hydrogen-bond network in the vicinity of Trp for the two Ans. The water molecules revealed in the proximity of the Trp residue have semiquantitative correlation with the observed emission spectral variations of (2,7-aza)Trp between AnsA and AnsB. Titration of aspartate, a competitive inhibitor of Ans, revealed an increase in N(7)-H emission intensity in AnsA but no obvious spectral changes in AnsB. The changes in the emission profiles reflect the modulation of structural states by locally confined environment and trapped-water collective motions.


Assuntos
Asparaginase/química , Triptofano/química , Asparaginase/metabolismo , Biocatálise , Isoenzimas/química , Isoenzimas/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Água/química
3.
J Phys Chem B ; 119(6): 2157-67, 2015 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-25046564

RESUMO

In this study, we used the tryptophan analogue, (2,7-aza)Trp, which exhibits water catalyzed proton transfer isomerization among N(1)-H, N(7)-H, and N(2)-H isomers, to probe the water environment of tryptophan-59 (Trp59) near the connecting loop region of ribonuclease Tl (RNase T1) by replacing the tryptophan with (2,7-aza)Trp. The resulting (2,7-aza)Trp59 triple emission bands and their associated relaxation dynamics, together with relevant data of 7-azatryptophan and molecular dynamics (MD) simulation, lead us to propose two Trp59 containing conformers in RNase T1, namely, the loop-close and loop-open forms. Water is rich in the loop-open form around the proximity of (2,7-aza)Trp59, which catalyzes (2,7-aza)Trp59 proton transfer in the excited state, giving both N(1)-H and N(7)-H isomer emissions. The existence of N(2)-H isomer in the loop-open form, supported by the MD simulation, is mainly due to the specific hydrogen bonding between N(2)-H proton and water molecule that bridges N(2)-H and the amide oxygen of Pro60, forming a strong network. The loop-close form is relatively tight in space, which squeezes water molecules out of the interface of α-helix and ß2 strand, joined by the connecting loop region; accordingly, the water-scant environment leads to the sole existence of the N(1)-H isomer emission. MD simulation also points out that the Trp-water pairs appear to preferentially participate in a hydrogen bond network incorporating polar amino acid moieties on the protein surface and bulk waters, providing the structural dynamic features of the connecting loop region in RNase T1.


Assuntos
Ribonuclease T1/química , Água/química , Substituição de Aminoácidos , Aspergillus oryzae/enzimologia , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Ribonuclease T1/genética , Triptofano
4.
Anal Biochem ; 437(1): 20-8, 2013 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-23462539

RESUMO

Human alpha-1-antitrypsin (A1PI) is a plasma protein with the function of protecting lung tissues from proteolytic destruction by enzymes from inflammatory cells. A1PI deficiency is an inherited disorder associated with pulmonary emphysema and a higher risk of chronic obstructive pulmonary disease (COPD). Here we present the structural characterization of a recombinant form of human A1PI (Hu-recA1PI) expressed in the human PER.C6 cell line using an array of analytical and biochemical techniques. Hu-recA1PI had the same primary structure as plasma-derived A1PI (pd-A1PI) except reduced N-terminal heterogeneity. The secondary and tertiary structures were indistinguishable from pd-A1PI. Like pd-A1PI, Hu-recA1PI was modified by N-linked glycosylation on N46, N83, and N246. Unlike pd-A1PI, most glycans on recA1P1 were core fucosylated via α(1-6) linkage. In addition, significantly higher amounts of tri- and tetraantennary glycans were observed. These differences in glycosylation contributed to the apparent higher molecular weight and lower isoelectric point (pI) of Hu-recA1PI than pd-A1PI. Hu-recA1PI contained both α(2-3)- and α(2-6)-linked sialic acids and had very similar sialylation levels as pd-A1PI. Hu-recA1PI glycans were differentially distributed, with N46 containing mostly biantennary glycans, N83 containing primarily tri- and tetraantennary glycans, and N247 containing exclusively biantennary glycans.


Assuntos
Proteínas Recombinantes/química , Proteínas Recombinantes/genética , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Fucose/metabolismo , Galactose/metabolismo , Expressão Gênica , Glicosilação , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Ácido N-Acetilneuramínico/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Retina/citologia , Especificidade por Substrato , alfa 1-Antitripsina/isolamento & purificação , alfa 1-Antitripsina/metabolismo
5.
J Lipid Res ; 47(1): 115-22, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16222033

RESUMO

Human plasma HDLs are classified on the basis of apolipoprotein composition into those that contain apolipoprotein A-I (apoA-I) without apoA-II [(A-I)HDL] and those containing apoA-I and apoA-II [(A-I/A-II)HDL]. ApoA-I enters the plasma as a component of discoidal particles, which are remodeled into spherical (A-I)HDL by LCAT. ApoA-II is secreted into the plasma either in the lipid-free form or as a component of discoidal high density lipoproteins containing apoA-II without apoA-I [(A-II)HDL]. As discoidal (A-II)HDL are poor substrates for LCAT, they are not converted into spherical (A-II)HDL. This study investigates the fate of apoA-II when it enters the plasma. Lipid-free apoA-II and apoA-II-containing discoidal reconstituted HDL [(A-II)rHDL] were injected intravenously into New Zealand White rabbits, a species that is deficient in apoA-II. In both cases, the apoA-II was rapidly and quantitatively incorporated into spherical (A-I)HDL to form spherical (A-I/A-II)HDL. These particles were comparable in size and composition to the (A-I/A-II)HDL in human plasma. Injection of lipid-free apoA-II and discoidal (A-II)rHDL was also accompanied by triglyceride enrichment of the endogenous (A-I)HDL and VLDL as well as the newly formed (A-I/A-II)HDL. We conclude that, irrespective of the form in which apoA-II enters the plasma, it is rapidly incorporated into spherical HDLs that also contain apoA-I to form (A-I/A-II)HDL.


Assuntos
Apolipoproteína A-II/sangue , Apolipoproteína A-I/sangue , Lipoproteínas HDL/sangue , Lipoproteínas HDL/química , Animais , Apolipoproteína A-II/administração & dosagem , Humanos , Injeções Intravenosas , Lipídeos/sangue , Lipoproteínas HDL/administração & dosagem , Masculino , Coelhos , Triglicerídeos/sangue
6.
J Biol Chem ; 278(25): 22530-6, 2003 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-12690114

RESUMO

The high density lipoproteins (HDL) in human plasma are classified on the basis of apolipoprotein composition into those containing apolipoprotein (apo) A-I but not apoA-II, (A-I)HDL, and those containing both apoA-I and apoA-II, (A-I/A-II)HDL. Cholesteryl ester transfer protein (CETP) transfers core lipids between HDL and other lipoproteins. It also remodels (A-I)HDL into large and small particles in a process that generates lipid-poor, pre-beta-migrating apoA-I. Lipid-poor apoA-I is the initial acceptor of cellular cholesterol and phospholipids in reverse cholesterol transport. The aim of this study is to determine whether lipid-poor apoA-I is also formed when (A-I/A-II)rHDL are remodeled by CETP. Spherical reconstituted HDL that were identical in size had comparable lipid/apolipoprotein ratios and either contained apoA-I only, (A-I)rHDL, or (A-I/A-II)rHDL were incubated for 0-24 h with CETP and Intralipid(R). At 6 h, the apoA-I content of the (A-I)rHDL had decreased by 25% and there was a concomitant formation of lipid-poor apoA-I. By 24 h, all of the (A-I)rHDL were remodeled into large and small particles. CETP remodeled approximately 32% (A-I/A-II)rHDL into small but not large particles. Lipid-poor apoA-I did not dissociate from the (A-I/A-II)rHDL. The reasons for these differences were investigated. The binding of monoclonal antibodies to three epitopes in the C-terminal domain of apoA-I was decreased in (A-I/A-II)rHDL compared with (A-I)rHDL. When the (A-I/A-II)rHDL were incubated with Gdn-HCl at pH 8.0, the apoA-I unfolded by 15% compared with 100% for the apoA-I in (A-I)rHDL. When these incubations were repeated at pH 4.0 and 2.0, the apoA-I in the (A-I)rHDL and the (A-I/A-II)rHDL unfolded completely. These results are consistent with salt bridges between apoA-II and the C-terminal domain of apoA-I, enhancing the stability of apoA-I in (A-I/A-II)rHDL and possibly contributing to the reduced remodeling and absence of lipid poor apoA-I in the (A-I/A-II)rHDL incubations.


Assuntos
Apolipoproteína A-II/farmacologia , Apolipoproteína A-I/deficiência , Glicoproteínas , Lipoproteínas HDL/metabolismo , Anticorpos Monoclonais , Apolipoproteína A-I/biossíntese , Apolipoproteína A-I/química , Proteínas de Transporte/sangue , Proteínas de Transporte/farmacologia , Proteínas de Transferência de Ésteres de Colesterol , Eletroforese em Gel de Poliacrilamida , Emulsões Gordurosas Intravenosas/farmacologia , Humanos , Cinética , Ligação Proteica , Desnaturação Proteica , Proteínas Recombinantes/farmacologia
7.
Microbiology (Reading) ; 146 Pt 12: 3141-3147, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11101672

RESUMO

A new protease inhibitor was purified to apparent homogeneity from a culture medium of Photorhabdus luminescens by ammonium sulfate precipitation and preparative isoelectric focusing followed by affinity chromatography. Ph. luminescens, a bacterium symbiotically associated with the insect-parasitic nematode Heterorhabditis bacteriophora, exists in two morphologically distinguishable phases (primary and secondary). It appears that only the secondary-phase bacterium produces this protease inhibitor. The protease inhibitor has an M:(r) of approximately 12000 as determined by SDS-PAGE. Its activity is stable over a pH range of 3.5-11 and at temperatures below 50 degrees C. The N-terminal 16 amino acids of the protease inhibitor were determined as STGIVTFKND(X)GEDIV and have a very high sequence homology with the N-terminal region of an endogenous inhibitor (IA-1) from the fruiting bodies of an edible mushroom, Pleurotus ostreatus. The purified protease inhibitor inactivated the homologous protease with an almost 1:1 stoichiometry. It also inhibited proteases from a related insect-nematode-symbiotic bacterium, Xenorhabdus nematophila. Interestingly, when present at a molar ratio of 5 to 1, this new protease inhibitor completely inactivated the activity of both trypsin and elastase. The activity of proteinase A and cathepsin G was partially inhibited by this bacterial protease inhibitor, but it had no effect on chymotrypsin, subtilisin, thermolysin and cathepsins B and D. The newly isolated protease inhibitor from the secondary-phase bacteria and its specific inhibition of its own protease provides an explanation as to why previous investigators failed to detect the presence of protease activity in the secondary-phase bacteria. The functional implications of the protease inhibitor are also discussed in relation to the physiology of nematode-symbiotic bacteria.


Assuntos
Endopeptidases/metabolismo , Photorhabdus/metabolismo , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/metabolismo , Rhabditoidea/microbiologia , Sequência de Aminoácidos , Animais , Temperatura Alta , Concentração de Íons de Hidrogênio , Insetos/parasitologia , Dados de Sequência Molecular , Peso Molecular , Photorhabdus/enzimologia , Photorhabdus/patogenicidade , Inibidores de Proteases/química , Simbiose , Xenorhabdus/enzimologia
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