Selective purification and characterization of adiponectin multimer species from human plasma

Adiponectin is an adipocyte-derived hormone and known to form several species of multimer, however, the precise components of each multimer have not been fully determined. We purified each multimer adiponectin selectively from human plasma and characterized them by affinity columns using anti-adiponectin, gelatin, or anti-albumin antibody and gel filtration. We found that adiponectin exists as four species of multimers in human plasma. According to their migrating mobility and N-terminal amino acid analysis, we defined them as a trimer, albumin-binding trimer, hexamer, and HMW. Low pH shifted HMW to hexamer, raising the possibility that HMW is a 12 mer or larger multimer. We also showed that HMW had the highest binding activity to the membrane fractions of C2C12 myocytes and activated AMPK most potently. Our results indicate that adiponectin forms diverse multimer species and at least some of the functional properties are dependent on a multimer status.     

High-molecular weight adiponectin isoforms increase after biliopancreatic diversion in obese subjects

Objective: Our objective was to test the effect of biliopancreatic diversion (BDP) in adiponectin multimerization. Adiponectin, the major protein secreted by adipose tissue, circulates in plasma in different isoforms. The most clinically relevant oligomers are high‐molecular weight (HMW) multimers and low‐molecular weight (LMW) trimers. Contrasting data on the effect of weight loss on adiponectin isoforms have been reported. Research Methods and Procedures: We measured total plasma adiponectin and HMW and LMW adiponectin oligomers (by Western blot analysis) before and 1 month after BPD, in 18 severely obese subjects. Results: One month after BPD, body weight decreased ～11%. Total adiponectin showed significant increase after BPD. In addition, we found a significant increase in HMW (percentage) adiponectin oligomers. We found a significant inverse correlation between HMW (percentage) and BMI before and after BPD. Homeostasis model of assessment‐insulin resistance decreased significantly after the BPD, without any significant correlation with total serum adiponectin and adiponectin oligomers. Discussion: A moderate weight loss after BPD increases total and HMW adiponectin oligomers. The significant correlation between BMI and HMW (percentage) adiponectin oligomers but not between BMI and total adiponectin might indicate a role of body fat mass in regulation of adiponectin multimerization. These data suggest that HMW oligomers represent a very sensitive parameter to short‐term BMI changes after BPD.     

Control of antibody high and low molecular weight species by depth filtration-based cell culture harvesting

Depth filtration-based harvesting is widely used in mAb manufacturing to remove cell and process-related impurities. However, it has not been studied on control of product-related impurities, which are very critical for product quality. In this article, we studied the interactions of depth filter with high and low molecular weight species (HMWs and LMWs) for their direct removal from cell culture. The process parameters (filter, loading, temperature, and flux) were evaluated for adsorption of HMWs and LMWs by depth filters. The adsorption is significantly dependent on filter media and loading capacity and is mainly on the basis of hydrophobic interaction during harvesting. The HMW and LMW species were characterized as HMW1, HMW2, LMW1, and LMW2. The increasing binding from LMW2 to LMW1, HMW1, and HMW2 is correlated with their increasing hydrophobicity score. Adsorption using enriched HMW sample demonstrated similar total protein binding capacity (36–40 g/m²) between depth filters D0HC and X0HC. However, X0HC has stronger HMW binding than D0HC (71% vs 43% of bound protein), indicating more hydrophobic interaction in X0HC. HMW2 DBC on X0HC reached 12 g/m², similar to protein binding on hydrophobic interaction membrane adsorbers. Further study showed LMW can induce HMW formation. This study provides a critical understanding of HMW and LMW interaction with depth filters. The strategy of HMW and LMW control by depth filtration-based harvesting was implemented successfully in mAb manufacturing.     

Degradation of proteins upon storage at near-neutral pH: Indications of a proteolytic/gelatinolytic activity associated with aggregates

Background

The twin phenomena of aggregation and degradation are classically associated with protein storage. However, although aggregation has been thought to be a possible consequence of protein degradation, it has never before been proposed to be a cause of degradation.

Methods

Proteins stored under physiological conditions and electrophoresed on SDS-PAGE were examined zymographically for the presence of detergent-resistant high molecular weight (HMW) forms, and association of such HMW forms with time-correlated, seeding-dependent gelatinolytic activity, under various conditions.

Results

Eight different proteins aggregate naturally during storage at near-neutral pH, with concomitant development of ‘gelatinolytic’ activity diminished greatly by storage at low temperatures, extremes of pH, arginine, imidazole, BSA, azide, EDTA, DTT, PMSF (but not AEBSF), and diisopropyl fluorophosphate (DFP), suggesting involvement of surface serine residues in a novel aggregate-borne proteolytic activity.

Conclusions

Naturally-formed aggregates of proteins appear to use surface serines to perform peptide bond hydrolysis, explaining degradation of proteins during storage, and indicating why aggregates are cytotoxic.

General significance

The study suggests that a bi-directional cause–effect relationship operates between protein aggregation, and protein degradation, providing clues to the design of better conditions for long-term protein storage.     

Adiponectin multimerization is dependent on conserved lysines in the collagenous domain: evidence for regulation of multimerization by alterations in posttranslational modifications

Adiponectin is a secreted, multimeric protein with insulin-sensitizing, antiatherogenic, and antiinflammatory properties. Serum adiponectin consists of trimer, hexamer, and larger high-molecular-weight (HMW) multimers, and these HMW multimers appear to be the more bioactive forms. Multimer composition of adiponectin appears to be regulated; however, the molecular mechanisms involved are unknown. We hypothesize that regulation of adiponectin multimerization and secretion occurs via changes in posttranslational modifications (PTMs). Although a structural role for intertrimer disulfide bonds in the formation of hexamers and HMW multimers is established, the role of other PTMs is unknown. PTMs identified in murine and bovine adiponectin include hydroxylation of multiple conserved proline and lysine residues and glycosylation of hydroxylysines. By mass spectrometry, we confirmed the presence of these PTMs in human adiponectin and identified three additional hydroxylations on Pro71, Pro76, and Pro95. We also investigated the role of the five modified lysines in multimer formation and secretion of recombinant human adiponectin expressed in mammalian cell lines. Mutation of modified lysines in the collagenous domain prevented formation of HMW multimers, whereas a pharmacological inhibitor of prolyl- and lysyl-hydroxylases, 2,2'-dipyridyl, inhibited formation of hexamers and HMW multimers. Bacterially expressed human adiponectin displayed a complete lack of differentially modified isoforms and failed to form bona fide trimers and larger multimers. Finally, glucose-induced increases in HMW multimer production from human adipose explants correlated with changes in the two-dimensional electrophoresis profile of adiponectin isoforms. Collectively, these data suggest that adiponectin multimer composition is affected by changes in PTM in response to physiological factors.     

Switching between the Alternative Structures and Functions of a 2-Cys Peroxiredoxin,by Site-Directed Mutagenesis

Members of the typical 2-Cys peroxiredoxin (Prx) subfamily represent an intriguing example of protein moonlighting behavior since this enzyme shifts function: indeed, upon chemical stimuli, such as oxidative stress, Prx undergoes a switch from peroxidase to molecular chaperone, associated to a change in quaternary structure from dimers/decamers to higher-molecular-weight (HMW) species. In order to detail the structural mechanism of this switch at molecular level, we have designed and expressed mutants of peroxiredoxin I from Schistosoma mansoni (SmPrxI) with constitutive HMW assembly and molecular chaperone activity. By a combination of X-ray crystallography, transmission electron microscopy and functional experiments, we defined the structural events responsible for the moonlighting behavior of 2-Cys Prx and we demonstrated that acidification is coupled to local structural variations localized at the active site and a change in oligomerization to HMW forms, similar to those induced by oxidative stress. Moreover, we suggest that the binding site of the unfolded polypeptide is at least in part contributed by the hydrophobic surface exposed by the unfolding of the active site. We also find an inverse correlation between the extent of ring stacking and molecular chaperone activity that is explained assuming that the binding occurs at the extremities of the nanotube, and the longer the nanotube is, the lesser the ratio binding sites/molecular mass is.     

High molecular weight renin identified in cytosol fractions of mouse renal cortices

In an attempt to confirm that high molecular weight renin was indeed true renin, we used a specific renin antibody and high performance liquid chromatography to determine characteristics of this protein. In mouse renin granules, renin was stored in a low molecular weight form of 38,000 daltons (LMW renin) and this molecular weight remained unchanged with application 20 mM of sodium tetrathionate. In the cytosol fraction of the renal cortex, LMW renin was partially converted to high molecular weight renin (HMW renin) of 65,000 daltons, as determined using tetrathionate. In both the LMW and HMW renin, enzymatic activity was completely neutralized by application of a specific antiserum of renin and an absolute amount of renin was identified by direct radioimmunoassay. The Km values of HMW and LMW renin were similar. Thus, LMW renin probably binds with renin binding substance and forms HMW renin.     

Transglutaminase-mediated fibronectin multimerization in lung endothelial matrix in response to TNF-alpha

Exposure of lung endothelial monolayers to tumor necrosis factor (TNF)-alpha causes a rearrangement of the fibrillar fibronectin (FN) extracellular matrix and an increase in protein permeability. Using calf pulmonary artery endothelial cell layers, we determined whether these changes were mediated by FN multimerization due to enhanced transglutaminase activity after TNF-alpha (200 U/ml) for 18 h. Western blot analysis indicated that TNF-alpha decreased the amount of monomeric FN detected under reducing conditions. Analysis of (125)I-FN incorporation into the extracellular matrix confirmed a twofold increase in high molecular mass (HMW) FN multimers stable under reducing conditions (P < 0.05). Enhanced formation of such HMW FN multimers was associated with increased cell surface transglutaminase activity (P < 0.05). Calf pulmonary artery endothelial cells pretreated with TNF-alpha also formed nonreducible HMW multimers of FN when layered on surfaces precoated with FN. Inhibitors of transglutaminase blocked the TNF-alpha-induced formation of nonreducible HMW multimers of FN but did not prevent either disruption of the FN matrix or the increase in monolayer permeability. Thus increased cell surface transglutaminase after TNF-alpha exposure initiates the enhanced formation of nonreducible HMW FN multimers but did not cause either the disruption of the FN matrix or the increase in endothelial monolayer permeability.     

乌拉尔图小麦(Triticum urartu)1Ay高分子量麦谷蛋白亚基基因编码区的克隆与鉴定(英文)

应用简并性引物和基因组PCR反应从乌拉尔图小麦(Triticum urartu)不同种质材料中获得并测定了表达型和沉默型1Ay高分子量麦谷蛋白亚基基因全长编码区的基因组DNA序列。表达型1Ay基因编码区的序列与前人已发表的y型高分子量麦谷蛋白亚基基因编码区的序列高度同源,由其推导的1Ay亚基的一级结构与已知的高分子量麦谷蛋白亚基相似。在细菌细胞中,表达型1Ay基因编码区的克隆序列可经诱导而产生1Ay蛋白,该蛋白与种子中1Ay亚基在电泳迁移率和抗原性上类似,表明所克隆的序列真实地代表了表达型1Ay基因的全长编码区。但是,本研究所克隆的沉默型1Ay基因的编码区序列因含有3个提前终止子而不能翻译成完整的1Ay蛋白。讨论了表达型1Ay基因在小麦籽粒加工品质改良中的潜在利用价值以及1Ay基因沉默的机制。     

小麦高分子量谷蛋白亚基及其基因的研究进展

主要介绍了小麦高分子量谷蛋白亚基（HMW－GS）及其基因的研究进展情况,目前,转基因小麦的技术已经逐渐成熟,由于分子生物学领域分子标记技术的迅速发展,尤其是PCR技术的广泛应用,为实现外源优良储藏蛋白基因导入改良品种提供了可能,利用已知小麦品种的基因序列设计引物,从众多的未知小麦品种中扩增出新基因加以研究并做外源优质HMW－GS基因的转入已成为一种趋势。     

从植物细胞核分离大分子量核DNA

研究了从植物中分离百万碱基对级大分子量核DNA的方法。该方法利用差速离心分离植物细胞核,经低熔点琼脂糖块或低熔点琼脂糖微珠包埋,蛋白酶K原位裂解后制备大分子量核DNA。结果表明,选择不同生长时期的材料和不同的包埋细胞核方式对大分子量核DNA的制备有很大的影响,由黄化苗或幼嫩的绿叶为材料分离细胞核,进行胶块包埋是制备大分子量核DNA的最佳条件。利用该法获得的DNA分子量在200kb－5．7Mb之间,主要集中在2．2～5．7Mb之间;每一胶块DNAE量为18～20μg。与包埋原生质体制备大分子量核DNA的方法相比,该方法获得的DNA纯度较高,去除了大部分细胞器DNA的污染;易于被限制性内切酶部分和完全消化,其消化结果具可重复性。该方法操作简单、适用植物种类广泛,用该方法从水稻（OryzasativaL．）、苹果（MaluspumilaMill．）、大豆（Glycinemax（L．）Merr．）、玉米（ZeamaysL．）等多种植物材料中成功地制备了大分子量核DNA。该方法制备的核DNA适用于植物的脉冲交变电泳基因组分析和构建人工细菌染色体文库和人工酵母染色体文库。     

乌拉尔图小麦(Triticum urartu)1Ay高分子量麦谷蛋白亚基基因编码区的克隆与鉴定

应用简并性引物和基因组PCR反应从乌拉尔图小麦(Triticum urartu)不同种质材料中获得并测定了表达型和沉默型1Ay高分子量麦谷蛋白亚基基因全长编码区的基因组DNA序列.表达型1Ay基因编码区的序列与前人已发表的y型高分子量麦谷蛋白亚基基因编码区的序列高度同源,由其推导的1Ay亚基的一级结构与已知的高分子量麦谷蛋白亚基相似.在细菌细胞中,表达型1Ay基因编码区的克隆序列可经诱导而产生1Ay蛋白,该蛋白与种子中1Ay亚基在电泳迁移率和抗原性上类似,表明所克隆的序列真实地代表了表达型1Ay基因的全长编码区.但是,本研究所克隆的沉默型1Av基因的编码区序列因含有3个提前终止子而不能翻译成完整的1Ay蛋白.讨论了表达型1Ay基因在小麦籽粒加工品质改良中的潜在利用价值以及lAy基因沉默的机制.     

Effect of dietary fenugreek seeds on biliary proteins that influence nucleation of cholesterol crystals in bile

Formation of cholesterol gallstones in gallbladder is controlled by procrystallising and anticrystallising factors present in bile. Dietary fenugreek seed has been recently observed to possess anti-lithogenic potential in experimental mice. In the current animal study, we evaluated the effect of dietary fenugreek on the compositional changes in the bile, particularly its effect on glycoproteins, low-molecular-weight (LMW) and high-molecular-weight (HMW) proteins, cholesterol nucleation time and cholesterol crystal growth. Groups of Wistar rats were fed for 10 weeks with diets: (1) basal control (C), (2) C + fenugreek (12%), (3) high cholesterol diet (HCD) and (4) HCD + fenugreek (12%). Feeding of HCD containing 0.5% cholesterol for 10 weeks rendered the bile lithogenic. Incorporation of fenugreek into HCD decreased the cholesterol content (70.5%), total protein (58.3%), glycoprotein (27.5%), lipid peroxides (13.6%) and cholesterol saturation index (from 1.98 to 0.75) in bile, increased the bile flow rate (19.5%), prolonged the cholesterol nucleation time and reduced the vesicular form of cholesterol (65%), which was accompanied with an increase in smaller vesicular form (94%). There was an increase in biliary phospholipid (33%) and total bile acid (49%) contents in the HCD + fenugreek group as compared with the HCD group. Electrophoretic separation of biliary LMW proteins showed the presence of a high concentration of 28-kDa protein, which might be responsible for the prolongation of cholesterol nucleation time in the fenugreek-fed groups. These findings indicate that the beneficial anti-lithogenic effect of dietary fenugreek, which primarily is due to reduction in the cholesterol content in bile, was additionally affected through a modulation of the nucleating and anti-nucleating proteins, which, in turn, affect cholesterol crystallisation.     

Cloning and characterization of two plasmids from Bacillus thuringiensis in Bacillus subtilis

Bacillus thuringiensis subspecies israliensis plasmids pTX14-1 and pTX14-3 were cloned and analyzed by Southern blot hybridization for their replication mechanism in Bacillus subtilis. The cloning of pTX14-1 into the replicon deficient vector pBOE335 showed the usual characteristics of single-stranded DNA plasmids, i.e., it generated circular single-stranded DNA and high molecular weight (HMW) multimers. The other plasmid, pTX14-3, behaved differently; it generated neither single-stranded DNA nor HMW multimers. Treatment with rifampicin did not result in the accumulation of single-stranded DNA. However, deletion of an EcoRI-PstI fragment resulted in the accumulation of both single-stranded DNA and HMW multimers. From various deletion derivatives, we have mapped the minus origin and the locus responsible for suppression of HMW multimer formation. Full activity of the minus origin and of the locus suppressing HMW formation was only observed on the native replicon, indicating a coupling to the plus strand synthesis.     

Impaired intracellular transport produced by a subset of type IIA von Willebrand disease mutations.

Type IIA von Willebrand disease (vWD) results from abnormalities in von Willebrand factor (vWF) characterized by absence of plasma high molecular weight (HMW) vWF multimers. In this report, 5 distinct point mutations were identified in 6 Type IIA vWD families. A total of 7 mutations, all clustered within a 124-amino acid segment of the vWF A2 domain, now account for 9 of a panel of 11 Type IIA families. In COS-7 cells, 3 single amino acid substitutions, Val844----Asp, Ser743----Leu, and Gly742----Arg, impaired the transport of vWF multimers between the endoplasmic reticulum and the Golgi complex, with more profound effects on the secretion of HMW multimers than lower molecular weight forms. In contrast, 2 substitutions, Arg834----Trp and Gly742----Glu, resulted in secretion of HMW multimers similar to wild-type vWF. The vWF structure observed within patient platelets correlated closely with the synthesis pattern seen for the corresponding mutants in COS-7 cells. These findings demonstrate that structural alterations within the A2 domain of vWF can produce the characteristic phenotype of Type IIA vWD via two distinct molecular mechanisms.     

Cloning and characterization of two plasmids from Bacillus thuringiensis in Bacillus subtilis

Bacillus thuringiensis subspecies israliensis plasmids pTX14-1 and pTX14-3 were cloned and analyzed by Southern blot hybridization for their replication mechanism in Bacillus subtilis. The cloning of pTX14-1 into the replicon deficient vector pBOE335 showed the usual characteristics of single-stranded DNA plasmids, i.e., it generated circular single-stranded DNA and high molecular weight (HMW) multimers. The other plasmid, pTX14-3, behaved differently; it generated neither single-stranded DNA nor HMW multimers. Treatment with rifampicin did not result in the accumulation of single-stranded DNA. However, deletion of an EcoRI-PstI fragment resulted in the accumulation of both single-stranded DNA and HMW multimers. From various deletion derivatives, we have mapped the minus origin and the locus responsible for suppression of HMW multimer formation. Full activity of the minus origin and of the locus suppressing HMW formation was only observed on the native replicon, indicating a coupling to the plus strand synthesis.     

带芒草属物种新型高分子量谷蛋白亚基的鉴定

采用SDSPAGE方法对牧草带芒草属3个种8份材料的高分子量谷蛋白进行了检测和鉴定。结果显示,带芒草物种具有的高分子量谷蛋白亚基与普通小麦中发现的不一样,其迁移率存在较大差异。其中,x型亚基均比Dx2亚基迁移率小或接近,y型亚基均比Dx12亚基迁移率大。8份材料中共发现了4种x型亚基新类型(Tax1,Tax2,Tax3和Tax4),5种y型亚基新类型(Tay1,Tay2,Tay3,Tay4和Tay5)和6种亚基组合类型(Tax1+Tay3,Tax3+Tay2,Tax4+Tay1,Tax1+Tay1,Tax2+Tay5,Tax4+Tay2),该项研究结果揭示了带芒草属植物可能具有与普通小麦类似的高分子量谷蛋白亚基,这些亚基在小麦品质遗传改良中具有潜在的利用价值。     

Size and shape of the repetitive domain of high molecular weight wheat gluten proteins. II. Hydrodynamic studies

This study describes the hydrodynamic properties of the repetitive domain of high molecular weight (HMW) wheat proteins, which complement the small-angle scattering (SANS) experiments performed in the first paper of this series. The sedimentation coefficients, s(0), and diffusion coefficients, D(0), were obtained from the homologous HMW proteins dB1 and dB4 that were cloned from the gluten protein HMW Dx5, and expressed in Escherichia coli. Monodisperse conditions for accurate determination of s(0) and D(0), were obtained by screening a series of buffers using dynamic light scattering. For the first time, hydrodynamic parameters were obtained on monodisperse samples that enabled the determination of the monomeric size and shape. The hydrodynamic values determined on dB1 and dB4 were used to test the worm-like chain (WLC) model that was proposed in the SANS studies. The successful matching of two separately obtained hydrodynamic parameters of dB1 and dB4 using the WLC model provides further evidence for the WLC model. The small discrepancy between the hydrodynamic and scattering data, possibly coming from the excluded volume effect, was compensated by a solvation layer of 1-2 water molecules thick around the protein in the WLC model. The solvation of the central domain is much higher than those of the terminal domains of the HMW subunits. This difference emphasizes the dual role of HMW wheat gluten proteins in water-binding and aggregation.     

Isolation and characterization of Glu-1 genes from the St genome of Pseudoroegneria libanotica

The St genome, which is present in nearly half of all Triticeae species, originates from the genus Pseudoroegneria. However, very little is known about the high molecular weight (HMW) subunits of glutenin which are encoded by the St genome. In this paper, we report the isolation from Pd. libanotica of four sequences encoding HMW subunits of glutenin. The four genes were all small compared to standard glutenin genes. All four sequences resemble y-type glutenins rather than x-types. However, their N-terminal domains contain a glutamine residue which is present in all x-type, but very few y-type subunits, and their central repetitive domains included some irregular motifs. The indication is therefore that the Glu-1St genes evolved earlier than other modern day homoeologues, so that they represent an intermediate state in the divergence between x- and y-type subunits. No x-type Glu-1St subunit genes were identified.     

Gastric mucosal injury activates bFGF gene expression and triggers preferential translation of high molecular weight bFGF isoforms through CUG-initiated, non-canonical codons

Basic fibroblast growth factor (bFGF or FGF-2) is a pleiotropic growth factor that promotes growth of mesenchymal and epithelial cells, stimulates angiogenesis and neuroprotection. Moreover, exogenous bFGF by stimulating angiogenesis promotes healing of gastroduodenal ulcers and cardiac and brain injury. All these actions were demonstrated in regard to 18 kDa bFGF isoform that is secreted by cells via an ER/Golgi-independent pathway and activates FGF receptors. However in some transformed and stressed cells and in some tissues (e.g. brain) the single copy bFGF gene encodes multiple gene products: 18 kDa and also higher molecular weight (HMW) bFGF isoforms: ∼21 and ∼22 kDa in rodents, and ∼22, ∼23 and ∼24 kDa in humans. The biologic roles of these HMW bFGF isoforms in vivo remain unknown. In this study we demonstrated that in normal, uninjured gastric mucosa, bFGF is almost exclusively expressed as 18 kDa isoform translated through a classical AUG (methionine) codon. In contrast, in injured gastric mucosa of rat, bFGF gene is preferentially translated to HMW bFGF isoforms through alternative CUG (leucine) initiation codon. Gastric mucosal injury caused in rats a significant increase in bFGF mRNA at 8 and 24 h vs. normal mucosa and a significant increase in bFGF protein at 24–72 h, mainly due to increased expression of ∼21 and ∼22 kDa HMW bFGF isoforms. This is first demonstration that gastric mucosal injury and repair triggers local activation of bFGF gene with preferential translation of HMW bFGF isoforms through a non-canonical CUG codon. This study uncovered CUG-initiated HMW bFGF translation as a novel regulatory mechanism operating in vivo during gastric injury repair.