Expression and characterization of recombinant human ciliary neurotrophic factor from Escherichia coli

The gene for ciliary neurotrophic factor (CNTF) was cloned from a human genomic DNA library by screening with a DNA fragment amplified from human genomic DNA using the polymerase chain reaction. A DNA sequence coding for human CNTF was placed under control of an regulatable promoter in the expression vector pJU1003 and transformed into Escherichia coli strain BL21(DE3). Induction of expression in cultures of this transformant led to the accumulation of approx. 25 mg/l per A600 unit of human CNTF. CNTF was purified to homogeneity from cell lysates via anion-exchange, cation-exchange and Zn(2+)-affinity chromatography. Purified CNTF contained less than 0.1% contaminating E. coli proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis and reversed-phase high-pressure liquid chromatography (HPLC). The protein exhibited an ultraviolet absorption maximum at 279 nm with a calculated extinction coefficient of A1%(279) = 9.0. Peptide map and amino acid sequence analyses confirmed that the expressed protein has the amino acid sequence expected for human CNTF, except for the absence of the amino-terminal methionine. High-purified recombinant human CNTF supported the survival of chick embryo parasympathetic, sympathetic and sensory neurons in culture at low picomolar concentrations. These results indicate that the biological activities previously ascribed to impure CNTF preparations indeed reside in one molecule.     

Cloning and expression of human ciliary neurotrophic factor

Ciliary neurotrophic factor (CNTF) is a survival factor for avian ciliary ganglion neurons and a variety of other neuronal cell types in vitro. We report here the cloning of the entire genomic sequence encoding human CNTF and its primary structure. Biologically active CNTF has been expressed in Chinese hamster ovary cells from a human genomic DNA clone. Human CNTF has no significant sequence similarity to any previously reported protein, although approximately 84% similarity exists compared with rat and rabbit CNTF. The lack of both an N-terminal signal sequence and consensus sequences for glycosylation or hydrophobic regions, and the fact that active CNTF is expressed but not released into the culture medium of transfected cells, argue in favour of human CNTF as a cytosolic protein. These data provide a basis for understanding the role of CNTF in nervous system physiology and pathology.     

Structure-function studies of human ciliary neurotrophic factor

Ciliary neurotrophic factor (CNTF) is a polypeptide that promotes the survival and/or differentiation of a number of neural cell types. Here we present a structural and functional analysis of the human CNTF molecule. Variant proteins were synthesized byEscherichia coli trnsformmed with mutant cDNA constructs, and purified by SDS-polyacrylamide gel electrophoresis and reverse phase high pressure liquid chromatography. Most variant CNTF proteins lacked neurotrophic activity, but two N-and C-terminal deletions (2–14 and 173–200, respectively) actually displayed a several-fold increase in specific activity. Loss of biological activity was accompanied by changes in the alphahelical nature of CNTF as measured by circular dichroism. These data strengthen the proposed similarity between CNTF and the family of hematopoietic cytokines.     

Overexpression of ciliary neurotrophic factor in vivo rescues chick ciliary ganglion neurons from cell death

Ciliary ganglion (CG) neurons undergo target-dependent cell death during embryonic development. Although ciliary neurotrophic factor (CNTF) was identified in vitro by its ability to support the survival of chick CG neurons, its function as a target-derived neurotrophic factor has been questioned by those working on mammalian-derived forms of CNTF. We have purified and cloned a chicken CNTF [chCNTF; formerly growth-promoting activity (GPA)] that is expressed in CG targets during the period of cell death and is secreted by cells transfected with chCNTF. In the present study we used a retroviral vector, RCASBP(A), to overexpress chCNTF in CG target tissues. Elevation of chCNTF biological activity three- to fourfold in the embryonic eye rescued an average of 31% of the neurons that would have normally died in vivo. In some individuals, nearly all of the neurons were rescued. ChCNTF had no effect on the number of neurons observed prior to cell death, nor were there any deleterious effects of either viral infection or overexpression of CNTF. These results show that chCNTF is able to function in vivo as a trophic factor for CG neurons, and suggest that limited availability of trophic support is one of the factors regulating CG neuron survival during development. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 283–293, 1998     

Synthesis and localization of ciliary neurotrophic factor in the sciatic nerve of the adult rat after lesion and during regeneration

Ciliary neurotrophic factor (CNTF) is expressed in high quantities in Schwann cells of peripheral nerves during postnatal development of the rat. The absence of a hydrophobic leader sequence and the immunohistochemical localization of CNTF within the cytoplasm of these cells indicate that the factor might not be available to responsive neurons under physiological conditions. However, CNTF supports the survival of a variety of embryonic neurons, including spinal motoneurons in culture. Moreover we have recently demonstrated that the exogenous application of CNTF protein to the lesioned facial nerve of the newborn rat rescued these motoneurons from cell death. These results indicate that CNTF might indeed play a major role in assisting the survival of lesioned neurons in the adult peripheral nervous system. Here we demonstrate that the CNTF mRNA and protein levels and the manner in which they are regulated are compatible with such a function in lesioned peripheral neurons. In particular, immunohistochemical analysis showed significant quantities of CNTF at extracellular sites after sciatic nerve lesion. Western blots and determination of CNTF biological activity of the same nerve segments indicate that extracellular CNTF seems to be biologically active. After nerve lesion CNTF mRNA levels were reduced to less than 5% in distal regions of the sciatic nerve whereas CNTF bioactivity decreased to only one third of the original before-lesion levels. A gradual reincrease in Schwann cells occurred concomitant with regeneration.     

Release of ciliary neurotrophic factor from cultured astrocytes and its modulation by cytokines

CNTF rescues various types of lesioned neurons in vivo, and it needs to be released from astrocytes into the extracellular space to have the effect. However, direct evidence for CNTF release has not been unequivocally demonstrated. We hypothesized that the rapid sequestration by CNTF receptor present on cultured astrocytes might be the cause of the inability to detect CNTF released into astrocyte-conditioned medium (ACM). Therefore, we measured CNTF immunoreactivity in medium conditioned by astrocytes treated with phosphatidylinositol-specific phospholipase C (PI-PLC) which was used to prevent released CNTF from binding to the CNTF receptor, since PI-PLC cleaves glycosyl-phosphatidylinositol anchor of CNTFR, the unique component involved in CNTF binding. CNTF was not detectable in untreated ACM, but was detectable in PI-PLC-treated ACM. These results together with the evidence that PI-PLC treatment did not have a toxic effect on astrocytes prove the fact that CNTF can be released from astrocytes without cell lysis. Subsequently, the effect of cytokines such as IL-1, TNF-, and EGF on CNTF release was examined. These cytokines increased CNTF protein levels in ACMs without increasing CNTF protein levels in astrocyte-extracts, indicating that they enhanced CNTF release from astrocytes.     

Preparation and biological properties of native and recombinant ciliary neurotrophic factor

CNTF (ciliary neurotrophic factor), purified from rabbit sciatic nerves by a relatively simple procedure, is bioactive in tissue culture at low picomolar concentration and appears as a doublet on polyacrylamide gel electrophoresis (PAGE). In these nerves, CNTF accounts for more than one-half of the survival-promoting activity on ciliary neurons. The concentration of CNTF in rabbit sciatic nerves is estimated to be 5 nmol/kg, more than 1000 times higher than would seem to be required to support neurons if the neurotrophic factor were homogeneously distributed. With recombinant DNA technology, rat CNTF has been synthesized in Escherichia coli, purified without denaturating agents, and found to be bioactive at a slightly lower concentration than CNTF extracted from rabbit sciatic nerves. After radioiodination, CNTF retains biological activity but is not specifically internalized and retrogradely transported in motor and sensory axons. In peripheral nerves, ciliary neurotrophic factor differs biologically from nerve growth factor (NGF) by its much higher tissue concentration and apparent lack of internalization by peripheral nerve axons. © 1992 John Wiley & Sons, Inc.     

睫状神经营养因子突变体蛋白的活性研究

为了进一步研究我室应用计算机分子模拟设计并表达纯化的睫状神经营养因子突变体蛋白的生物学活性,分别采用鸡胚背根神经节无血清培养法、TF-1细胞增殖法、正常小鼠减重法对其活性进行研究。结果是突变体蛋白能促进鸡胚背根神经节的生长;促进TF-1细胞增殖,MTT测定法表明突变体蛋白与国际参考品相比,比活不低于2.0×106U/mg;使正常小鼠的体重减轻,摄食量减少,脂肪指数下降,并且体重的减轻与突变体蛋白的给药剂量呈现良好的剂量依赖关系,其ED50为：150.986?g/kg/d。以上实验表明CNTF突变体蛋白具有促神经生长、促TF-1细胞增殖和减重的生物学活性。从而为其进一步的应用和开发提供了线索。     

睫状神经营养因子对大鼠去神经骨骼肌的营养作用

目的：了解睫状神经营养因子（CNTF）对去神经引起的肌肉萎缩的治疗作用。方法：离断SD大鼠一侧坐骨神经,连续给予CNTF20d,观察肌肉湿重、蛋白含量、肌纤维横截面积、收缩性能和残肢程度。结果：①给予0.2mg/kg的CNTF,可使损务侧肌纤维横截面积增加35％,肌肉湿重增加38％,胫前肌总蛋白含量增加24％,腓长肌强直收缩强度提高40％,显著改善肢残程度;②0.2mg/kg的CNTF作用明显强于0.05mg/kg的CNTF;③此目鱼肌（慢肌）比伸趾长肌（快肌）对CNTF更敏感。结论：CNTF能显著改善成年大鼠坐骨神经离断后骨骼肌的萎缩和功能丧失,该效应的强弱与用药剂量和肌肉类型有关。     

Isolation and characterization of phosphofructokinase C from rabbit brain

Phosphofructokinase from rabbit brain consists of hybrids of the A, B, and C isozymes. Phosphofructokinase C was isolated from a purified mixture of such hybrids in a 2-step procedure. In the first step, phosphofructokinase B was removed by chromatography on DEAE-Sephadex. In the second step, subunits of phosphofructokinases A and C were separated by dissociation at pH 5.0 followed by chromatography on carboxymethylcellulose. The separated isozymes were then reassociated by neutralization. Phosphofructokinase C was structurally distinct from phosphofructokinases A (obtained from muscle or brain) and B (obtained from liver) as shown by one-dimensional chymotryptic and staphylococcal V8 protease fingerprints of all three isozymes. In addition, phosphofructokinase C cross-reacted weakly or not at all with antisera raised against phosphofructokinase B or phosphofructokinase A. Phosphofructokinase C was also kinetically distinct from the A and B isozymes. The C isozyme was more sensitive than the A isozyme but less sensitive than the B isozyme to inhibition by ATP, was less sensitive than the A isozyme but more sensitive than the B isozyme to inhibition by citrate, and was less sensitive than either of the other two isozymes to activation by inorganic phosphate, AMP, and fructose 2,6-bisphosphate. The self-association properties of phosphofructokinase C differed from those of the A and B isozymes in that at pH 8.0, the C isozyme did not form oligomers larger than a tetramer under conditions where the other two isozymes did. Thus the properties of phosphofructokinase C are in general quite distinct from those of the other two phosphofructokinase isozymes.     

Isolation and characterization of cathepsin B from rabbit testis

Cathepsin B (EC 3.4.22.1) has been purified from rabbit testes to apparent homogeneity by chromatography on DE-52, affinity chromatography on organomercurial agarose and subsequent gel filtrations on Sephadex G-75. The enzyme is composed of a single polypeptide of Mr 23,000. Thiol blocking agents and leupeptin abolished the activity of the enzyme completely. The enzyme showed maximum activity at pH 6.0 and 43 degrees C, required 2 mM-cysteine for the optimal activity and had a Km1.45 X 10(-3) M using Z-Arg-beta-naphthylamide as the substrate. However, Z-Arg-Arg-beta-naphthylamide was 12 times more sensitive as a substrate than was Z-Arg-beta-naphthylamide. Rabbit testicular cathepsin B hydrolysed intact proteins. An endogenous inhibitor isolated from the rabbit testes inhibited purified Cathepsin B.     

Isolation and characterization of L-fucose dehydrogenase from rabbit liver

L-Fucose dehydrogenase [EC 1.1.1.122] was isolated from a rabbit liver extract and purified about 390-fold with a yield of approximately 13%. The purification procedures included treatment with protamine, ammonium sulfate fractionation, treatment with acid, DE-32 celluose colum chromatography, gel filtration on Sephadex G-100, preparative polyacrylamide gel electrophoresis, and affinity chromatography on 5' AMP-Sepharose 4B. The last procedure, affinity chromatography on 5' AMP-Sephadex 4B, was useful for the removal of other dehydrogenases. The eznyme which was homogeneous, as shown by polyacrylamide gel electrophoresis, had a molecular weight of about 92,000. The optimum pH was at 10.0 and isoelectric point at 5.2. The enzyme accepted both L-fucose and D-arabinose as substrate, but was specific for NAD+ as coenzyme. Km values were 0.15 mM, 1.4 mM, and 0.7 mM for L-fucose, D-arabinose, and NAD+, respectively. A single enzyme catalyzed the oxidation of L-fucose and D-arabinose, which had the same configurations of hydroxyl groups from C-2 to C-4. The reaction products obtained with L-fucose as substrate were L-fucono-lactone and L-fuconic acid. The L-fucono-lactone was an immediate product of oxidation and was hydrolyzed to L-fuconic acid spontaneously. This reaction was irreversible. Therefore, it is likely that L-fucose dehydrogenase is involved in the initial step of the catabolic pathway of L-fucose in rabbit liver.     

Membrane-associated neurotransmitter stimulating factor is very similar to ciliary neurotrophic factor.

Membrane-associated neurotransmitter stimulating factor (MANS) can modulate sympathetic neurotransmitter expression and promote ciliary neuron survival in cell culture. Previous studies have shown that its biological effects and biochemical properties are similar to those of ciliary neurotrophic factor (CNTF). In addition, CNTF is present in spinal cord, the source of MANS. These observations raised the possibility that MANS preparations contain CNTF. We find that partially purified MANS fractions contain a 24-kD protein that is recognized in Western blots by an antiserum generated against recombinant rat CNTF (rCNTF). This antiserum immunoprecipitates virtually all the cholinergic-inducing and the ciliary neurotrophic activities present in MANS preparations. When iodinated rCNTF is incubated with spinal cord membranes, a significant proportion of the labeled CNTF segregates with the membrane pellet. The membrane-associated exogenous CNTF can be eluted from the membrane fraction by treatment with high-salt solutions, similar to that used to solubilize MANS from spinal cord membranes. Our data suggest that a substantial portion of the cholinergic differentiation and ciliary neurotrophic activities present in MANS preparations can be attributed to CNTF or a CNTF-like molecule.     

Preparation and biological properties of native and recombinant ciliary neurotrophic factor.

CNTF (ciliary neurotrophic factor), purified from rabbit sciatic nerves by a relatively simple procedure, is bioactive in tissue culture at low picomolar concentration and appears as a doublet on polyacrylamide gel electrophoresis (PAGE). In these nerves, CNTF accounts for more than one-half of the survival-promoting activity on ciliary neurons. The concentration of CNTF in rabbit sciatic nerves is estimated to be 5 nmol/kg, more than 1000 times higher than would seem to be required to support neurons if the neurotrophic factor were homogeneously distributed. With recombinant DNA technology, rat CNTF has been synthesized in Escherichia coli, purified without denaturating agents, and found to be bioactive at a slightly lower concentration than CNTF extracted from rabbit sciatic nerves. After radioiodination, CNTF retains biological activity but is not specifically internalized and retrogradely transported in motor and sensory axons. In peripheral nerves, ciliary neurotrophic factor differs biologically from nerve growth factor (NGF) by its much higher tissue concentration and apparent lack of internalization by peripheral nerve axons.     

Morphological effects of ciliary neurotrophic factor treatment during neuromuscular synapse elimination

In adult skeletal muscles, exogenous ciliary neurotrophic factor (CNTF) induces axons and their nerve terminals to sprout. CNTF also regulates the amount of multiple innervation in developing skeletal muscles during synapse elimination, maintaining multiple innervation of muscle fibers. While CNTF may maintain multiple innervation by regulating developmental synapse elimination, it is also possible that CNTF induces the formation of new multiple innervation through a sprouting response. In this study I examined morphologically the effects of CNTF during synapse elimination in the extensor digitorum longus (EDL) muscle. Rat pups received injections of CNTF in one leg and vehicle in the other either early [postnatal day 7 (P7)-P13] or late (P14–P20) in development. The early treatment period corresponds to that time when the pattern of innervation in the EDL is converted from predominantly multiple to single innervation. The late treatment period is at the end of synapse elimination for the EDL but corresponds to the major period of synapse elimination in the levator ani (LA), allowing a comparison of effects on these two muscles from the same animals. On the day after the final injection, EDL muscles were dissected and stained with tetranitroblue tetrazolium and the resulting pattern of innervation was assessed. The present findings indicate that only the early CNTF treatment regulates the level of multiple innervation in the EDL. Moreover, the effect of early CNTF treatment was local, affecting multiple innervation only in the EDL from the CNTF-treated leg. CNTF injected during the late treatment period had no apparent effects on the EDL but had a potent effect on the pattern of innervation in the LA, significantly increasing the level of multiple innervation in this muscle. Thus, CNTF affected multiple innervation in these two muscles only if provided during the period when single innervation normally develops. There was no evidence to indicate that CNTF induced axons or their terminals to sprout during either treatment period. In conclusion, CNTF increases the level of multiple innervation, probably by regulating synapse elimination, and skeletal muscles themselves may be an important target site for CNTF action. Presumably, the sprouting response to CNTF found in adult muscle develops sometime after P21. © 1996 John Wiley & Sons, Inc.     

Isolation and characterization of a mannan-binding protein from rabbit serum

A binding protein which recognizes mannose and N-acetylglucosamine has been isolated from rabbit serum to apparent homogeneity. The serum binding protein was nearly identical to the mannan-binding protein isolated previously from rabbit liver [Kawasaki, T., Etoh, R. and Yamashina, I. (1978) Biochem. Biophys. Res. Commun. $\text{81}$, 1018–1024] in respect of immunochemical properties and subunit profiles, but could be differentiated from the liver protein in its larger molecular size and inferior sensitivity to monosaccharides as haptenic inhibitors of the binding to ¹²⁵I-mannan. A postulation was made that the plasma was, comparable with the liver, a major locus of mannan-binding protein in the rabbit.