Different isoforms of the soluble leptin receptor in non-pregnant and pregnant mice

Leptin circulates in murine serum in a free and a bound form. As shown in humans, a soluble leptin receptor (sOB-R), which modulates the effects of its ligand, circulates in murine blood. The aim of our study was to determine abundance and biochemical nature of this protein. For the quantification of sOB-R we developed a ligand-immunofunctional assay (LIFA) which is based on both, leptin binding and immunological recognition. The use of this LIFA revealed that during late gestation sera of pregnant mice had a approximately 290-fold higher level of sOB-R than non-pregnant animals. As investigated by size exclusion chromatography these mice sera demonstrated a co-elution of their leptin binding activity with leptin immunoreactivity and levels of sOB-R measured by LIFA. Therefore, it has to be concluded that sOB-R represents the major leptin binding activity in murine circulation. The molecular analysis of sOB-R by Western blot and by cross-linking with 125I-leptin in sera of pregnant and non-pregnant mice demonstrated two different isoforms of sOB-R, which were capable of leptin binding. The sOB-R in serum migrated at a molecular weight of 150kDa in pregnant and only of 120kDa in non-pregnant animals. Deglycosylation of these isoforms led to sOB-R molecules which were found at the same molecular weight in SDS-PAGE. This finding indicates that both isoforms differ only in the degree of their glycosylation. In conclusion, the non-pregnant and the pregnant states are accompanied by differently glycosylated isoforms of sOB-R whose physiological relevance remains to be determined.     

Contribution of leptin receptor N-linked glycans to leptin binding

The extracellular domain of the human leptin receptor (Ob-R) contains 20 potential N-glycosylation sites whose role in leptin binding remains to be elucidated. We found that a mammalian cell-expressed sOb-R (soluble Ob-R) fragment (residues 22-839 of the extracellular domain) bound leptin with a dissociation constant of 1.8 nM. This binding was inhibited by Con A (concanavalin A) or wheatgerm agglutinin. Treatment of sOb-R with peptide N-glycosidase F reduced leptin binding by approximately 80% concurrently with N-linked glycan removal. The human megakaryoblastic cell line, MEG-01, expresses two forms of the Ob-R, of approx. 170 and 130 kDa molecular mass. Endo H (endoglycosidase H) treatment and cell culture with alpha-glucosidase inhibitors demonstrated that N-linked glycans are of the complex mature type in the 170 kDa form and of the high-mannose type in the 130 kDa form. Both isoforms bound leptin, but not after peptide N-glycosidase F treatment. An insect-cell-expressed sOb-R fragment, consisting of the Ig (immunoglobulin), CRH2 (second cytokine receptor homology) and FNIII (fibronectin type III) domains, bound leptin with affinity similar to that of the entire extracellular domain, but this function was abolished after N-linked glycan removal. The same treatment had no effect on the leptin-binding activity of the isolated CRH2 domain. Our findings show that N-linked glycans within Ig and/or FNIII domains regulate Ob-R function, but are not involved in essential interactions with the ligand.     

人Leptin基因的cDNA的克隆和表达

克隆人Leptin基因的cDNA并获取表达的Leptin蛋白,为进一步研究新的Leptin相关蛋白打下基础。以人基因组DNA为模板,用引物悬挂延伸PCR法,克隆与6个组氨酸（6×His）密码子相连的人Leptin基因的cDNA,并将其克隆到体外表达载体pIVEX23MCS上,通过体外快速翻译系统(Roche公司的RTS500环状模板试剂盒和RTS500 ProteoMaster仪器)在体外表达了带有6×His的Leptin融合蛋白。经SDSPAGE及Western blot方法鉴定,融合蛋白大小正确,有172个氨基酸,分子量为1946kD,具有特异的抗原性,且主要以可溶形式存在于反应液上清中。     

人可溶性低密度脂蛋白受体在甲醇酵母中的表达

为获得低密度脂蛋白受体配基结合结构域在甲醇酵母中的分泌表达 ,首先用RT PCR方法以人肝癌Bel 740 2总RNA为模板扩增了编码低密度脂蛋白受体配基结合结构域的基因片段。核酸测序分析表明克隆到的DNA片段的序列与报道的人LDLR的cDNA序列相同。然后构建了甲醇酵母表达质粒pPIC9K sLDLr ,并将其线性化后用电穿孔法导入PichiapastorisGS115。分别用SDS PAGE、Westernblot和Ligandbindingblot对GS115 pPIC9K sLDLr上清中的重组sLDLR进行鉴定。SDS PAGE和Westernblot分析表明表达的sLDLR的表观分子量为 36kD。Ligandbindingblot分析表明表达的sLDLR具有配基结合的生物学活性     

Subcellular localization and internalization of the four human leptin receptor isoforms.

There are four known isoforms of the human leptin receptor (HLR) with different C-terminal cytoplasmic domains (designated by the number of unique C-terminal amino acids). In cells expressing HLR-5, -15, or -274, 15-25% of the leptin binding sites were located at the plasma membrane. In contrast, in cells expressing HLR-67, only 5% of the total binding sites were at the plasma membrane. Immunofluorescent microscopy showed that all four isoforms partially co-localized with calnexin and beta-COP, markers of the endoplasmic reticulum and the Golgi, respectively. All isoforms were also detected in an unidentified punctate compartment. All isoforms were internalized via clathrin-mediated endocytosis, but at different rates. After 20 min at 37 degrees C, 45% of a bound cohort of labeled ligand had been internalized by HLR-15, 30% by HLR-67, 25% by HLR-274, and 15% by HLR-5. Degradation of internalized leptin occurred in lysosomes. Overnight exposure to leptin down-regulated all isoforms, but to a variable extent. HLR-274 displayed the greatest down-regulation and also appeared to reach lysosomes more quickly than the other isoforms. The faster degradation of HLR-274 may help to terminate leptin signaling.     

Comparison of Membrane Surface Proteins of Sperm Cells and Somatic Protoplasts of Zea mays

Viable sperm cells and somatic protoplasts (leaf, callus) of Zea mays were successfully isolated and purified. The plasma membrane surface proteins of intact somatic protoplasts and sperm cells were compared after probing with N-hydroxysuccinimido-biotin (NHS-bi-otin). Horseradish peroxidase-labelled avidin (HRP-avidin) was used to detect membrane proteins after separation by SDS-PAGE and Western blot. Four protein bands characteristic of the surface membrane of sperm cells were identified varying from 48 to 78 kD, five bands of leaf protoplasts in the range of 45~78 kD, and two bands of callus protoplasts, 67 and 80 kD were detected. One protein of 48 kD was specific to the surface membrane of sperm cells and might be related to the specific roles of sperm cell physiology.     

Purification and characterization of placental protein 5

This report describes the purification of placental protein 5, PP5, from the human placenta by two affinity chromatography steps, the first with Heparin-Sepharose and the second with Sepharose-linked monoclonal anti-PP5 antibody. The final purification is achieved by reversed-phase high performance liquid chromatography. In SDS-polyacrylamide gel electrophoresis under reducing or nonreducing conditions, PP5 purified in this study migrates as one major band at 36 kD. The previously purified PP5 is more heterogeneous: under nonreducing conditions it migrates at 30 kD and, after reduction, it gives three bands at 16.8 kD, 18.3 kD, and 19.0 kD. In Western blot analysis, both purified proteins react with polyclonal and monoclonal anti-PP5 antibodies. Three N-terminal amino acid sequences are obtained for the previously purified PP5, whereas the N-terminal of PP5 purified in this study is blocked. These results suggest that PP5 previously purified in the absence of protease inhibitors, does not represent the native form of PP5. Computer comparison of the obtained amino acid sequences revealed no significant homology to known protein sequences.     

肾综合征出血热纯化疫苗的SDS-PAGE分析

为了证明蛑综合征出血热纯化疫苗的主要成分坦病毒蛋白,采用出血热纯化疫苗经浓缩后进行SDS－PAGE和Western-blotting分析。结果 经SDS－PAGE显示,肾综合征出血热纯化疫苗有三条蛋白带,分子量分别约为70kD、55kD和50kD,与汉坦病毒三种结构蛋白（糖蛋白G1、G2和核蛋白NP）的分子量相符;经Western-blotting显示,分子量50kD的蛋白带反应阳性,分子量70kD和55kD的蛋白带无反应,认定出血热纯化疫苗的主要成分为汉坦病毒蛋白,主要由G1、G2和NP三种结构蛋白构成。     

鼻咽癌组织p53相关蛋白的纯化

鼻咽癌是南方常见的恶性肿瘤,ＮＰＣ与ＥＢ病毒关系十分密切。通过免疫亲和层析的方法了解ＮＰＣ肿瘤标本中ｐ５３与病毒或细胞蛋白间潜在的相互作用。这种相互作用可能引起Ｐ５３在ＮＰＣ组织中累积。建立单克隆抗体ｐＡｂ１８０１,ｐＡｂ２４０１免疫亲和层析柱,从ＮＰＣ转移淋巴结分离ｐ５３结合蛋白。     

Mammalian single-stranded DNA binding proteins and heterogeneous nuclear RNA proteins have common antigenic determinants.

Antibodies were raised in rabbit against a pure subset of calf thymus single-stranded DNA binding proteins (ssDBPs) and purified by affinity chromatography on antigen-Sepharose. In Western blot experiments these antibodies were shown to react to the same extent with the whole family of bovine ssDBPs, as well as with ssDBPs from HeLa cells. When used to stain total cell extracts from both calf thymus and HeLa cells the antibodies reacted only with bands corresponding to the ssDBPs and with a set of bands of higher molecular weight, whose electrophoretic pattern matched that of the 40S hnRNP core proteins. In effect we observed that purified 40S hnRNP core proteins from HeLa cells were strongly reactive with the antibodies. Moreover after partial tryptic digestion HeLa cells ssDBPs and hnRNPs produced immunoreactive fragments of the same molecular weight and isoelectric point. Extensive structural homologies can thus be evidenced between these two classes of proteins, which share the property of selective binding to single-stranded nucleic acids.     

Purification and partial amino acid sequence analysis of two distinct tumor necrosis factor receptors from HL60 cells

Two distinct tumor necrosis factor (TNF) receptors of 55- and 75-kDa apparent molecular masses previously identified on the cell surface by monoclonal antibodies have been solubilized with Triton X-100 from HL60 cells. A filter-based dot blot assay was developed to monitor specific 125I-TNF alpha binding during fractionation of the cell extract. By a combination of immuno- and ligand affinity chromatography and reverse phase high performance liquid chromatography both receptor proteins were purified to apparent homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two bands at 55 and 51 kDa for the 55-kDa TNF receptor and a major 75-kDa and a minor 65-kDa band for the 75-kDa TNF receptor. All these bands specifically bound TNF alpha and TNF beta in ligand blot experiments. The exclusive specificity of monoclonal antibodies of the utr series for the 75.65-kDa bands and of the htr series for the 55.51-kDa bands was demonstrated with the purified antigens on Western blots. Both TNF receptor types were found to contain N-linked carbohydrates. N-terminal amino acid sequence analysis of the 55- and 51-kDa bands of the 55-kDa TNF receptor revealed identical sequences suggesting a possible truncation at the C-terminal end. Two different N-terminal sequences were determined for the 65-kDa band. One corresponded to the published sequence of ubiquitin; the other was therefore assumed to be a unique sequence of the 75-kDa TNF receptor. Additional internal sequences of this receptor were determined after proteolytic cleavage.     

鸡胚骨骼肌组织M-CAT结合因子的初步鉴定

采用偶联ＣＡＴＴＧＣＴ寡核苷酸的ＤＮＡ亲和层析柱从发育１３ｄ鸡胚骨骼肌核抽提物中分离到两种核蛋白．ＳＤＳ－ＰＡＧＥ结果表明,被ＤＮＡ亲和柱滞留的两种核蛋白分子量分别为３０ｋＤ和３２ｋＤ．凝胶阻滞结合竞争分析显示,纯化的核蛋白可与Ｍ－ＣＡＴ共有序列ＣＡＴＴＣＣＴ特异结合．Ｓｏｕｔｈｗｅｓｔｅｒｎ印迹技术确定仅３０ｋＤ分子可直接识别、结合ＣＡＴＴＣＣＴ元件,但３２ｋＤ分子却不能．结果提示,３０ｋＤ分子为依赖ＤＮＡ的Ｍ－ＣＡＴ结合因子,３２ｋＤ分子属性有待进一步研究证实     

SARS病毒受体ACE2的克隆、原核表达及其功能区鉴定

ACE2(angiotensin-converting enzyme 2,ACE2)是SARS冠状病毒(severe acute respiratory syndrome associatedcoronavirus,SARS-CoV)的主要受体。此研究旨在鉴定ACE2的SARS-CoV受体功能区,为进一步阐明SARS-CoV与细胞间的相互作用机制及研制抗病毒药物等提供理论依据。利用RT-PCR从Vero-E6细胞的mRNA中分两段扩增ACE2基因,其中N端片段ACE2A1-367(102~1 210nt)不包括ACE2的酶活性位点(1 223~1 237nt,或374~378aa),而C端片段ACE2B335-805(1 101~2 524nt)包括ACE2的酶活性位点。扩增片段克隆入pMD-18T,并进行测序鉴定。进一步构建与GST基因融合表达的原核表达质粒pGEX-ACE2A与pGEX-ACE2B,IPTG诱导表达。表达的融合蛋白分子量为65kD和77kD,主要以包涵体形式存在。Western blot证实表达产物具有免疫学活性。将纯化的包涵体蛋白质复性后进行Western blot分析,证实pGEX-ACE2A表达的蛋白(~65kD)能与SARS-CoV S1蛋白特异结合,而pGEX-ACE2B表达的蛋白(~77kD)不能与S1蛋白结合。结果表明,ACE2的受体活性与酶活性位点无关,受体功能区在ACE2 N端367个氨基酸内。     

A rapid,quantitative immunofunctional assay for measuring human leptin

BACKGROUND: We have developed a rapid, sensitive and quantitative in vitro assay for leptin based upon its ability to bind to the soluble extracellular domain of the leptin receptor (sOB-R). Such an assay is theoretically capable of differentiating between physiologically active leptin molecules from those with modified, either enhanced or reduced, binding activity. METHODS: A preparation of sOB-R was immobilized to capture leptin from serum samples or standards. Anti-leptin antibodies that had been raised in rabbits were added in a second incubation step to identify leptin molecules bound to sOB-R. Signal detection was performed in a third incubation step by anti-rabbit IgG labeled with peroxidase. The immunofunctional assay (IFA) was clinically validated by the comparison of leptin levels in adolescents (n = 41, age range 9-18 years, BMI range 13.4-33.8 kg/m(2) and adults (n = 80, age range 18-77 years, BMI range 16.4-54.7 kg/m(2) measured using the IFA with data of an in-house RIA performed with the same standards and leptin antibodies. RESULTS: The functional sensitivity of the IFA was 0.4 ng/ml and comparable to the data of the RIA. Intra- and interassay coefficients of variation were below 12.5 % in both methods. Leptin levels correlated well with the BMI of the subjects studied (r = 0.70 for RIA, r = 0.72 for IFA; p < 0.0001) as well as between IFA (y) and RIA (x) (y = x -1.31 ng/ml; r = 0.97, p < 0.0001). The median of the quotient between IFA and RIA levels was 0.86 (quartile range 0.60-1.10) for all samples. CONCLUSIONS: So far, only at the most minor differences between leptin measurements using the newly developed IFA and those using a conventional RIA have been detected. Additional studies using the IFA method are required to investigate whether or not discrepant results with the IFA will be seen in various states of relative leptin resistance and whether or not such differences are of biological relevance.     

Afadin: A Novel Actin Filament–binding Protein with One PDZ Domain Localized at Cadherin-based Cell-to-Cell Adherens Junction

A novel actin filament (F-actin)–binding protein with a molecular mass of ~205 kD (p205), which was concentrated at cadherin-based cell-to-cell adherens junction (AJ), was isolated and characterized. p205 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. p205 was a protein of 1,829 amino acids (aa) with a calculated molecular mass of 207,667 kD. p205 had one F-actin–binding domain at 1,631–1,829 aa residues and one PDZ domain at 1,016– 1,100 aa residues, a domain known to interact with transmembrane proteins. p205 was copurified from rat brain with another protein with a molecular mass of 190 kD (p190). p190 was a protein of 1,663 aa with a calculated molecular mass of 188,971 kD. p190 was a splicing variant of p205 having one PDZ domain at 1,009–1,093 aa residues but lacking the F-actin–binding domain. Homology search analysis revealed that the aa sequence of p190 showed 90% identity over the entire sequence with the product of the AF-6 gene, which was found to be fused to the ALL-1 gene, known to be involved in acute leukemia. p190 is likely to be a rat counterpart of human AF-6 protein. p205 bound along the sides of F-actin but hardly showed the F-actin–cross-linking activity. Northern and Western blot analyses showed that p205 was ubiquitously expressed in all the rat tissues examined, whereas p190 was specifically expressed in brain. Immunofluorescence and immunoelectron microscopic studies revealed that p205 was concentrated at cadherin-based cell-to-cell AJ of various tissues. We named p205 l-afadin (a large splicing variant of AF-6 protein localized at adherens junction) and p190 s-afadin (a small splicing variant of l-afadin). These results suggest that l-afadin serves as a linker of the actin cytoskeleton to the plasma membrane at cell-to-cell AJ.     

Partial purification of microsomal signal peptidase from hen oviduct

Signal peptidase has been purified approximately 600-fold from hen oviduct microsomes. Treatment of microsomes with ice-cold sodium carbonate at pH 11.5 removes soluble and extrinsic membrane proteins prior to solubilization of signal peptidase with Nonidet P-40. After dialysis to pH 8.2, the solubilized enzyme is chromatographed on diethylaminoethyl cellulose at pH 8.2. More than 90% of contaminating proteins bind to the column while signal peptidase and endogenous phospholipid are eluted in the column void volume. Enzyme activity subsequently binds to carboxymethyl cellulose at pH 5.8 and is eluted by approximately 100 to 200 mM NaCl during a NaCl gradient. Polypeptides present in partially purified hen oviduct signal peptidase have relative molecular masses ranging from 54 kD to less than 11 kD with major bands at 29, 23, 22, 19, 18 and 13 kD. The purified peptidase requires phospholipid for activity and is maximally active in the presence of 2 mg/ml phosphatidylcholine.     

Effects of leptin to cultured growth plate chondrocytes

OBJECTIVE: This study aimed to evaluate whether leptin has any effect on growth plate chondrocytes. METHODS: We studied the effects of exogenous leptin on cultured rabbit growth plate chondrocytes. This involved assessing [3H]thymidine incorporation, alkaline phosphatase (ALP) activity, proteoglycan production, leptin receptor (Ob-R) activity, and detection of Ob-R using Western blot analysis. RESULTS: The existence of Ob-R in growth plate chondrocytes was revealed by Western blot and Ob-R activity. Prior to semiconfluence, leptin increased [3H]thymidine incorporation while at the semiconfluent and early confluent stages, leptin promoted ALP activity and tended to promote proteoglycan production. CONCLUSION: Growth plate chondrocytes possess Ob-Rs, and leptin enhance chondrocyte proliferation and subsequent cell differentiation.     

LAK细胞中一种自由基清除剂——SLP的分离纯化与抗体制备

ＳＯＤ样蛋白（ＳＯＤ－ｌｉｋｅ ｐｒｏｔｅｉｎ,ＳＬＰ）是从ＬＡＫ 细胞中发现的一种具有自由基清除功能的蛋白．为阐明ＳＬＰ的生化特性和确定其蛋白序列或基因序列,采用ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ ＦＦ层析从ＬＡＫ 细胞中得到部分纯化的ＳＬＰ,并采用ＩＥＦ等电聚焦电泳,活性染色,将含有活性蛋白的胶条直接免疫Ｂａｌｂ／ｃ 小鼠,制备抗血清并筛选得到多株有中和ＳＬＰ清除自由基活性的单克隆抗体．Ｗｅｓｔｅｒｎ ｂｌｏｔ结果表明ＳＬＰ分子量约为６７ ｋＤ,并测得ｐＩ约为４．２．     

Effects of body fat on the associations of high-molecular-weight adiponectin, leptin and soluble leptin receptor with metabolic syndrome in Chinese

Background

Little is known regarding the associations between high-molecular-weight (HMW-) adiponectin, leptin and soluble leptin receptor (sOB-R) and metabolic syndrome (MetS) in Chinese. Also few studies elucidate the effects of inflammation and body fat mass on the relations.

Methods

Plasma HMW-adiponectin, leptin and sOB-R were measured among 1055 Chinese men and women (35∼54 yrs). Whole body and trunk fat mass were determined by Dual-energy X-ray absorptiometry. MetS was defined by the updated NCEP/ATPIII criterion for Asian-Americans.

Results

HMW-adiponectin was inversely associated with MetS in multivariate model including fat mass index (FMI), inflammatory markers, leptin and sOB-R (OR in the highest quartile  = 0.30, 95%CI 0.18∼0.50, P<.0001). Plasma sOB-R was also inversely associated with MetS independent of body fatness and inflammatory markers, whereas the association was somewhat attenuated after adjusting HMW-adiponectin (OR for the highest quartile  = 0.78, 95%CI 0.47∼1.32, P = 0.15). In contrast, leptin was associated with increased odds of MetS independent of inflammatory markers, HMW-adiponectin, and sOB-R (OR for the highest quartile  = 2.64, 95%CI 1.35∼5.18, P = 0.006), although further adjustment for FMI abolished this association.

Conclusions

HMW-adiponectin exhibited strong inverse associations with MetS independent of body composition, inflammation, leptin and sOB-R; while the associations of leptin and sOB-R were largely explained by fat mass or HMW-adiponectin, respectively.     

犬瘟热病毒细胞膜受体的鉴定

犬瘟热病毒（ＣＤＶ）敏感细胞Ｖｅｒｏ用ＳＤＳ或ＲＩＰＡ溶解缓冲液溶解,利用病毒铺覆蛋白印迹技术（ＶＯＰＢＡ）鉴定犬瘟热病毒疫苗株（ＣＤＶ－ｏｎｄｅｓｔｅｐｏｏｒｔ）的细胞受体。结果发现,在Ｖｅｒｏ细胞上有两组ＣＤＶ结合蛋白质,即高分子量组蛋白质（１２７ｋＤ、１２０ｋＤ、１１０ｋＤ）与低分子量组蛋白质（２７ｋＤ和３０ｋＤ）。这些ＣＤＶ结合蛋白组分的性质及在ＣＤＶ致病中的作用有等进一步研究。