Role of disulfide bonds in biologic activity of human interleukin-6.

We have examined the functional importance of the two disulfide bonds formed by the four conserved cysteines of human interleukin (IL-6). Using a bacterial expression system, we have synthesized a series of recombinant IL-6 mutants in which the constituent cysteines of the first (Cys45-Cys51), second (Cys74-Cys84), or both disulfide bonds of recombinant human interleukin-6 were replaced by other amino acids. Each mutant was partially purified and tested in four representative bioassays. While mutants lacking Cys45 and Cys51 retained activity similar to nonmutated recombinant IL-6, the activity of mutants lacking Cys74 and Cys84 was significantly reduced, especially in assays involving human cell lines. These results indicate that the first disulfide bond of human interleukin-6 is not required for maintenance of normal biologic activity. However, the fact that mutants lacking Cys45 and Cys51 were more active than corresponding cysteine-free mutants indicates that the disulfide bond formed by these residues contributes to biologic activity in the absence of the second disulfide bond. Competition binding studies with representative mutants indicate that their affinity for the human IL-6 receptor parallels their biologic activities on human cells.     

Disulfide and secondary structures of recombinant human granulocyte colony stimulating factor

Molecular characteristics and secondary structures of recombinant methionyl human granulocyte colony stimulating factor produced by genetically engineered Escherichia coli are described. Limited radiolabeling of the protein with tritiated iodoacetate and determination of the labeled residue revealed that this recombinant protein contains only one free cysteine at position 17 which is not essential for activity. The free cysteine is inaccessible to modification unless the molecule is unfolded under denaturing conditions. The molecule forms two disulfide bridges which were assigned as Cys(36)-Cys(42) and Cys(64)-Cys(74) based on the results of isolation and characterization of disulfide-containing peptides obtained from a subtilisin digest of the intact protein. CD analyses and secondary structure prediction suggest that the molecule is abundant in alpha-helical structures.     

Folding and oxidation of recombinant human granulocyte colony stimulating factor produced in Escherichia coli. Characterization of the disulfide-reduced intermediates and cysteine----serine analogs.

The folding and oxidation of recombinant human granulocyte colony-stimulating factor solubilized from Escherichia coli inclusion bodies was investigated. During the folding process, two intermediates, I1 and I2, were detected by kinetic studies using high performance liquid chromatography. I1 exists transiently and disappears quickly with the concomitant formation of I2. In contrast, I2 requires a longer time to fold into the final oxidized form, N. CuSO4 catalysis increases the folding rate of I2 from I1, while CuSO4 and elevated temperature (37 degrees C) have a dramatic effect on the folding rate of N from I2. These observations suggest the following sequential oxidative folding pathway. [sequence: see text] Peptide map analysis of the iodoacetate-labeled intermediates revealed that I1 represents the fully reduced granulocyte colony-stimulating factor containing 5 free cysteines; I2 is the partially oxidized species containing a single Cys36-Cys42 disulfide bond; and N, the final folded form, has two disulfide bonds. The physicochemical properties and biological activities of I1, I2, N, and several Cys----Ser analogs made by site-directed mutagenesis were further investigated. In guanidine hydrochloride-induced denaturation studies, the disulfide-reduced intermediates and the analogs missing either of the disulfide bonds are conformationally less stable than those of the wild type molecule or the analog with the free Cys at position 17 changed to Ser. Recombinant human granulocyte colony stimulating factor lacking either disulfide bond or both has overall secondary and tertiary structures different from those of the wild type molecule and exhibits lower biological activity. These studies show that disulfide bond formation is crucial for maintaining the molecule in a properly folded and biologically active form.     

Disulfide bond structure and N-glycosylation sites of the extracellular domain of the human interleukin-6 receptor

The high affinity interleukin-6 (IL-6) receptor is a hexameric complex consisting of two molecules each of IL-6, IL-6 receptor (IL-6R), and the high affinity converter and signaling molecule, gp130. The extracellular "soluble" part of the IL-6R (sIL-6R) consists of three domains: an amino-terminal Ig-like domain and two fibronectin-type III (FN III) domains. The two FN III domains comprise the cytokine-binding domain defined by a set of 4 conserved cysteine residues and a WSXWS sequence motif. Here, we have determined the disulfide structure of the human sIL-6R by peptide mapping in the absence and presence of reducing agent. Mass spectrometric analysis of these peptides revealed four disulfide bonds and two free cysteines. The disulfides Cys102-Cys113 and Cys146-Cys157 are consistent with known cytokine-binding domain motifs, and Cys28-Cys77 with known Ig superfamily domains. An unusual cysteine connectivity between Cys6-Cys174, which links the Ig-like and NH2-terminal FN III domains causing them to fold back onto each other, has not previously been observed among cytokine receptors. The two free cysteines (Cys192 and Cys258) were detected as cysteinyl-cysteines, although a small proportion of Cys258 was reactive with the alkylating agent 4-vinylpyridine. Of the four potential N-glycosylation sites, carbohydrate moieties were identified on Asn36, Asn74, and Asn202, but not on Asn226.     

Determination of the disulfide structure and N-glycosylation sites of the extracellular domain of the human signal transducer gp130

gp130 is the common signal transducing receptor subunit for the interleukin-6-type family of cytokines. Its extracellular region (sgp130) is predicted to consist of five fibronectin type III-like domains and an NH2-terminal Ig-like domain. Domains 2 and 3 constitute the cytokine-binding region defined by a set of four conserved cysteines and a WSXWS motif, respectively. Here we determine the disulfide structure of human sgp130 by peptide mapping, in the absence and presence of reducing agent, in combination with Edman degradation and mass spectrometry. Of the 13 cysteines present, 10 form disulfide bonds, two are present as free cysteines (Cys(279) and Cys(469)), and one (Cys(397)) is modified by S-cysteinylation. Of the 11 potential N-glycosylation sites, Asn(21), Asn(61), Asn(109), Asn(135), Asn(205), Asn(357), Asn(361), Asn(531), and Asn(542) are glycosylated but not Asn(224) and Asn(368). The disulfide bonds, Cys(112)-Cys(122) and Cys(150)-Cys(160), are consistent with known cytokine-binding region motifs. Unlike granulocyte colony-stimulating factor receptor, the connectivities of the four cysteines in the NH2-terminal domain of gp130 (Cys(6)-Cys(32) and Cys(26)-Cys(81)) are consistent with known superfamily of Ig-like domains. An eight-residue loop in domain 5 is tethered by Cys(436)-Cys(444). We have created a model predicting that this loop maintains Cys(469) in a reduced form, available for ligand-induced intramolecular disulfide bond formation. Furthermore, we postulate that domain 5 may play a role in the disulfide-linked homodimerization and activation process of gp130.     

Conformational analysis of intermediates involved in the in vitro folding pathways of recombinant human macrophage colony stimulating factor beta by sulfhydryl group trapping and hydrogen/deuterium pulsed labeling

In vitro oxidative folding of reduced recombinant human macrophage colony stimulating factor beta (rhm-CSFbeta) involves two major events: disulfide isomerization in the monomeric intermediates and disulfide-mediated dimerization. Kinetic analysis of rhm-CSFbeta folding indicated that monomer isomerization is slower than dimerization and is, in fact, the rate-determining step. A time-dependent determination of the number of free cysteines remaining was made after refolding commence. The folding intermediates revealed that rhm-CSFbeta folds systematically, forming disulfide bonds via multiple pathways. Mass spectrometric evidence indicates that native as well as non-native intrasubunit disulfide bonds form in monomeric intermediates. Initial dimerization is assumed to involve formation of disulfide bonds, Cys 157/159-Cys' 157/159. Among six intrasubunit disulfide bonds, Cys 48-Cys 139 and Cys' 48-Cys' 139 are assumed to be the last to form, while Cys 31-Cys' 31 is the last intersubunit disulfide bond that forms. Conformational properties of the folding intermediates were probed by H/D exchange pulsed labeling, which showed the coexistence of noncompact dimeric and monomeric species at early stages of folding. As renaturation progresses, the noncompact dimer undergoes significant structural rearrangement, forming a native-like dimer while the monomer maintains a noncompact conformation.     

Identification of the disulfide bonds in human plasma protein SP-40,40 (apolipoprotein-J).

SP-40,40, a human plasma protein, is a modulator of the membrane attack complex formation of the complement system as well as a subcomponent of high-density lipoproteins. In the present study, the positions of the disulfide bonds in SP-40,40 were determined. SP-40,40 was purified from human seminal plasma by affinity chromatography using an anti-SP-40,40 monoclonal antibody and reversed-phase, high-performance liquid chromatography (HPLC). The protein was digested with trypsin and the fragments were separated by reversed-phase HPLC. The peptides containing disulfide bonds were fluorophotometrically detected with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The peptides containing more than two disulfide bonds were further digested with Staphylococcus aureus V8 protease and lysylendopeptidase, and the fragments were isolated by HPLC. The amino acid compositions and the amino acid sequences of the peptides containing only a disulfide bond were determined. Disulfide bonds thus determined were between Cys58(alpha)-Cys107(beta), Cys68(alpha)-Cys99(beta), Cys75(alpha)-Cys94(beta), and Cys86(alpha)-Cys80(beta). Since there was no free sulfhydryl groups in the SP-40,40 molecule, Cys78(alpha) and Cys91(beta) should also be linked by a disulfide bond. It is notable that all of the disulfide bonds in SP-40,40 are not only formed by inter-chain pairing, but also appear to form an antiparallel ladder-like structure between the two chains. The unique structure could be related to the functions of SP-40,40.     

Characterization of a recombinant murine interleukin-6: assignment of disulfide bonds

Murine interleukin 6 (mIL-6) has been synthesized as a fusion protein using a lac operon inducible plasmid in Escherichia coli. The first 8 amino acids are from the N-terminus of bacterial beta-galactosidase and the last 175 amino acids are from residue number 12 to the end of native mIL-6. This fusion protein is equipotent with the native molecule in the hybridoma growth factor assay and has comparable receptor binding characteristics. The two disulfide bridges in mIL-6 have been identified by Staphylococcus aureus V8 protease peptide mapping and Edman degradation of cystine-containing peptides. It has been shown that there are disulfide bonds between Cys46-Cys52 and Cys75-Cys85.     

Interleukin-2 signalling is modulated by a labile disulfide bond in the CD132 chain of its receptor

Certain disulfide bonds present in leucocyte membrane proteins are labile and can be reduced in inflammation. This can cause structural changes that result in downstream functional effects, for example, in integrin activation. Recent studies have shown that a wide range of membrane proteins have labile disulfide bonds including CD132, the common gamma chain of the receptors for several cytokines including interleukin-2 and interleukin-4 (IL-2 and IL-4). The Cys(183)-Cys(232) disulfide bond in mouse CD132 is susceptible to reduction by enzymes such as thioredoxin (TRX), gamma interferon-inducible lysosomal thiolreductase and protein disulfide isomerase, which are commonly secreted during immune activation. The Cys(183)-Cys(232) disulfide bond is also reduced in an in vivo lipopolysaccharide (LPS)-induced acute model of inflammation. Conditions that lead to the reduction of the Cys(183)-Cys(232) disulfide bond in CD132 inhibit proliferation of an IL-2-dependent T cell clone and concomitant inhibition of the STAT-5 signalling pathway. The same reducing conditions had no effect on the proliferation of an IL-2-independent T cell clone, nor did they reduce disulfide bonds in IL-2 itself. We postulate that reduction of the Cys(183)-Cys(232) disulfide in CD132 inhibits IL-2 binding to the receptor complex. Published data show that the Cys(183)-Cys(232) disulfide bond is exposed at the surface of CD132 and in close contact with IL-2 and IL-4 in their respective receptor complexes. In addition, mutants in these Cys residues in human CD132 lead to immunodeficiency and loss of IL-2 binding. These results have wider implications for the regulation of cytokine receptors in general, as their activity can be modulated by a 'redox regulator' mechanism caused by the changes in the redox environment that occur during inflammation and activation of the immune system.     

Identification of the disulfide bonds in the recombinant somatomedin B domain of human vitronectin

The NH(2)-terminal somatomedin B (SMB) domain (residues 1-44) of human vitronectin contains eight Cys residues organized into four disulfide bonds and is required for the binding of type 1 plasminogen activator inhibitor (PAI-1). In the present study, we map the four disulfide bonds in recombinant SMB (rSMB) and evaluate their functional importance. Active rSMB was purified from transformed Escherichia coli by immunoaffinity chromatography using a monoclonal antibody that recognizes a conformational epitope in SMB (monoclonal antibody 153). Plasmon surface resonance (BIAcore) and competitive enzyme-linked immunosorbent assays demonstrate that the purified rSMB domain and intact urea-activated vitronectin have similar PAI-1 binding activities. The individual disulfide linkages present in active rSMB were investigated by CNBr cleavage, partial reduction and S-alkylation, mass spectrometry, and protein sequencing. Two pairs of disulfide bonds at the NH(2)-terminal portion of active rSMB were identified as Cys(5)-Cys(9) and Cys(19)-Cys(21). Selective reduction/S-alkylation of these two disulfide linkages caused the complete loss of PAI-1 binding activity. The other two pairs of disulfide bonds in the COOH-terminal portion of rSMB were identified as Cys(25)-Cys(31) and Cys(32)-Cys(39) by protease-generated peptide mapping of partially reduced and S-alkylated rSMB. These results suggest a linear uncrossed pattern for the disulfide bond topology of rSMB that is distinct from the crossed pattern present in most small disulfide bond-rich proteins.     

Evidence for difference in the roles of two cysteine residues involved in disulfide bond formation in the folding of human lysozyme

Human lysozyme is made up of 130 amino acid residues and has four disulfide bonds at Cys6-Cys128, Cys30-Cys116, Cys65-Cys81, and Cys77-Cys95. Our previous results using the Saccharomyces cerevisiae secretion system indicate that the individual disulfide bonds of human lysozyme have different functions in the correct in vivo folding and enzymatic activity of the protein (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). In this paper, we report the results of experiments that were focused on the roles of Cys65 and Cys81 in the folding of human lysozyme protein in yeast. A mutant protein (C81A), in which Cys81 was replaced with Ala, had almost the same enzymatic activity and conformation as those of the native enzyme. On the other hand, another mutant (C65A), in which Cys65 was replaced with Ala, was not found to fold correctly. These results indicate that Cys81 is not a requisite for both correct folding and activity, whereas Cys65 is indispensable. The mutant protein C81A is seen to contain a new, non-native disulfide bond at Cys65-Cys77. The possible occurrence of disulfide bond interchange during our mapping experiments cannot be ruled out by the experimental techniques presently available, but characterization of other mutant proteins and computer analysis suggest that the intramolecular exchange of disulfide bonds is present in the folding pathway of human lysozyme in vivo.     

Localization of disulfide bonds in the cystine knot domain of human von Willebrand factor

von Willebrand factor (VWF) is a multimeric glycoprotein that is required for normal hemostasis. After translocation into the endoplasmic reticulum, proVWF subunits dimerize through disulfide bonds between their C-terminal cystine knot-like (CK) domains. CK domains are characterized by six conserved cysteines. Disulfide bonds between cysteines 2 and 5 and between cysteines 3 and 6 define a ring that is penetrated by a disulfide bond between cysteines 1 and 4. Dimerization often is mediated by additional cysteines that differ among CK domain subfamilies. When expressed in a baculovirus system, recombinant VWF CK domains (residues 1957-2050) were secreted as dimers that were converted to monomers by selective reduction and alkylation of three unconserved cysteine residues: Cys(2008), Cys(2010), and Cys(2048). By partial reduction and alkylation, chemical and proteolytic digestion, mass spectrometry, and amino acid sequencing, the remaining intrachain disulfide bonds were characterized: Cys(1961)-Cys(2011) (), Cys(1987)-Cys(2041) (), Cys(1991)-Cys(2043) (), and Cys(1976)-Cys(2025). The mutation C2008A or C2010A prevented dimerization, whereas the mutation C2048A did not. Symmetry considerations and molecular modeling based on the structure of transforming growth factor-beta suggest that one or three of residues Cys(2008), Cys(2010), and Cys(2048) in each subunit mediate the covalent dimerization of proVWF.     

Location of disulfide bonds within the sequence of human serum cholinesterase

Human serum cholinesterase was digested with pepsin under conditions which left disulfide bonds intact. Peptides were isolated by high pressure liquid chromatography, and those containing disulfide bonds were identified by a color assay. Peptides were characterized by amino acid sequencing and composition analysis. Human serum cholinesterase contains 8 half-cystines in each subunit of 574 amino acids. Six of these form three internal disulfide bridges: between Cys65-Cys92, Cys252-Cys263, and Cys400-Cys519. A disulfide bond with Cys65 rather than Cys66 was inferred by homology with Torpedo acetylcholinesterase. Cys571 forms a disulfide bridge with Cys571 of an identical subunit. This interchain disulfide bridge is four amino acids from the carboxyl terminus. A peptide containing the interchain disulfide is readily cleaved from cholinesterase by trypsin (Lockridge, O., and La Du, B. N. (1982) J. Biol. Chem. 257, 12012-12018), suggesting that the carboxyl terminus is near the surface of the globular tetrameric protein. The disulfide bridges in human cholinesterase have exactly the same location as in Torpedo californica acetylcholinesterase. There is one potential free sulfhydryl in human cholinesterase at Cys66, but this sulfhydryl could not be alkylated. Comparison of human cholinesterase, and Torpedo and Drosophila acetylcholinesterases to the serine proteases suggests that the cholinesterases constitute a separate family of serine esterases, distinct from the trypsin family and from subtilisin.     

Disulfide bonding arrangements in active forms of the somatomedin B domain of human vitronectin

The N-terminal cysteine-rich somatomedin B (SMB) domain (residues 1-44) of the human glycoprotein vitronectin contains the high-affinity binding sites for plasminogen activator inhibitor-1 (PAI-1) and the urokinase receptor (uPAR). We previously showed that the eight cysteine residues of recombinant SMB (rSMB) are organized into four disulfide bonds in a linear uncrossed pattern (Cys(5)-Cys(9), Cys(19)-Cys(21), Cys(25)-Cys(31), and Cys(32)-Cys(39)). In the present study, we use an alternative method to show that this disulfide bond arrangement remains a major preferred one in solution, and we determine the solution structure of the domain using NMR analysis. The solution structure shows that the four disulfide bonds are tightly packed in the center of the domain, replacing the traditional hydrophobic core expected for a globular protein. The few noncysteine hydrophobic side chains form a cluster on the outside of the domain, providing a distinctive binding surface for the physiological partners PAI-1 and uPAR. The hydrophobic surface consists mainly of side chains from the loop formed by the Cys(25)-Cys(31) disulfide bond, and is surrounded by conserved acidic and basic side chains, which are likely to contribute to the specificity of the intermolecular interactions of this domain. Interestingly, the overall fold of the molecule is compatible with several arrangements of the disulfide bonds. A number of different disulfide bond arrangements were able to satisfy the NMR restraints, and an extensive series of conformational energy calculations performed in explicit solvent confirmed that several disulfide bond arrangements have comparable stabilization energies. An experimental demonstration of the presence of alternative disulfide conformations in active rSMB is provided by the behavior of a mutant in which Asn(14) is replaced by Met. This mutant has the same PAI-1 binding activity as rVN1-51, but its fragmentation pattern following cyanogen bromide treatment is incompatible with the linear uncrossed disulfide arrangement. These results suggest that active forms of the SMB domain may have a number of allowed disulfide bond arrangements as long as the Cys(25)-Cys(31) disulfide bond is preserved.     

Structural and functional significance of disulfide bonds in saxatilin,a 7.7 kDa disintegrin

Saxatilin is a 7.7 kDa disintegrin that belongs to a family of homologous protein found in several snake venoms. Six disulfide bond locations of the disintegrin were determined by enzymatic cleavage and matrix-assisted-laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF). Functional implications of the disulfide bonds related to the biological activity of saxatilin were investigated with recombinant protein species produced by site-directed mutagenesis of saxatilin. Several lines of experimental evidence indicated that three disulfide bonds, Cys21-Cys35, Cys29-Cys59, and Cys47-Cys67, of the disintegrin are closely associated with its biological function such as its ability to block the binding of integrin GPIIb-IIIa and alpha(v)beta(3) with fibrinogen and extracellular matrix. Those disulfide linkages were also revealed to be important for maintaining the functional structure of the protein molecule. On the other hand, the disulfide bridges of Cys6-Cys15 and Cys8-Cys16 do not appear to be critical for the molecular structure and function of saxatilin.     

Elucidation of the disulfide bridge pattern of the recombinant human growth and differentiation factor 5 dimer and the interchain Cys/Ala mutant monomer

Growth and differentiation factor 5 (GDF5) is involved in many developmental processes such as chondrogenesis and joint and bone formation. A recombinant monomeric human GDF5 mutant rGDF5(C84A) is in vitro as potent as the dimeric native form, and clinical investigations of rGDF5(C84A) are in progress. Native homodimeric GDF5 belongs to the transforming growth factor β (TGF-β) superfamily; each monomer contains a cystine knot formed by three intrachain disulfide bridges, and the monomers are connected via an interchain disulfide bridge. The disulfide bridge pattern of recombinant homodimeric rGDF5 was recently elucidated by X-ray diffraction. A combination of proteolytic degradation with thermolysin, separation of the generated fragments by reverse-phase high-performance liquid chromatography (RP–HPLC), and subsequent analyses of the disulfide-linked peptides by electrospray–mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrometry, amino acid analysis, and Edman degradation led to the unambiguous identification of the disulfide bridge pattern of the monomeric mutant rGDF5(C84A) and of the homodimeric rGDF5 in solution. The cystine knot of homodimeric rGDF5 exhibits the pattern Cys1-Cys5, Cys2-Cys6, and Cys3-Cys7 (three intrachain disulfide bonds), and the monomers are connected by a single interchain disulfide bridge (Cys4-Cys4) in accordance with other members of the TGF-β superfamily. The monomeric mutant rGDF5(C84A) exhibits the same cystine knot pattern as homodimeric rGDF5.     

Defining the native disulfide topology in the somatomedin B domain of human vitronectin

The N-terminal 44 amino acid residues of the human plasma glycoprotein vitronectin, known as the somatomedin B (SMB) domain, mediates the interaction between vitronectin and plasminogen activator inhibitor 1 (PAI-1) in a variety of important biological processes. Despite the functional importance of the Cys-rich SMB domain, how its four disulfide bridges are arranged in the molecule remains highly controversial, as evidenced by three different disulfide connectivities reported by several laboratories. Using native chemical ligation and orthogonal protection of selected Cys residues, we chemically synthesized all three topological analogs of SMB with predefined disulfide connectivities corresponding to those previously published. In addition, we oxidatively folded a fully reduced SMB in aqueous solution, and prepared, by CNBr cleavage, the N-terminal segment of 51 amino acid residues of intact vitronectin purified from human blood. Proteolysis coupled with mass spectrometric analysis and functional characterization using a surface plasmon resonance based vitronectin-PAI-1-SMB competition assay allowed us to conclude that 1) only the Cys(5)-Cys(21), Cys(9)-Cys(39), Cys(19)-Cys(32), and Cys(25)-Cys(31) connectivity is present in native vitronectin; 2) only the native disulfide connectivity is functional; and 3) the native disulfide pairings can be readily formed during spontaneous (oxidative) folding of the SMB domain in vitro. Our results unequivocally define the native disulfide topology in the SMB domain of human vitronectin, providing biochemical as well as functional support to the structural findings on a recombinant SMB domain by Read and colleagues (Zhou, A., Huntington, J. A., Pannu, N. S., Carrell, R. W., and Read, R. J. (2003) Nat. Struct. Biol. 10, 541-544).     

Disulfide arrangement of human insulin-like growth factor I derived from yeast and plasma.

The disulfide arrangement of yeast derived human insulin-like growth factor I (yIGF-I) was determined using a combination of Staphylococcus aureus V8 protease mapping, fast-atom-bombardment mass spectrometry as well as amino acid sequence and composition analysis. Three disulfide bridges were found between the following cysteine residues: Cys6-Cys48, Cys47-Cys52 and Cys18-Cys61. IGF-I isolated from human plasma (pIGF-I) was found to have an identical disulfide configuration. A yeast-derived isomeric form of IGF-I (yisoIGF-I) exhibited an altered disulfide arrangement: Cys6-Cys47, Cys48-Cys52 and Cys18-Cys61. Radioreceptor analysis of pIGF-I and yIGF-I showed high specific activity, 20,000 U/mg. However, yisoIGF-I demonstrated a severely reduced ability to bind to the IGF-I receptor (19%) and was less potent in provoking a mitogenic response in Balb/C 3T3 fibroblasts (50% at doses 10-100 ng/ml). The data demonstrate the importance of correct disulfide arrangement in IGF-I for full biological activity.     

Production, purification and characterization of biologically active recombinant human nerve growth factor

The human NGF gene was isolated and inserted downstream from murine leukemia virus LTR in a plasmid having dihydrofolate reductase cDNA. The expression plasmid was introduced into CHO cells. Selection of the transformants for the resistance to methotrexate gave a CHO cell line which produced human NGF at a level of 4 mg/L in the culture medium. The recombinant human NGF was purified to near homogeneity from the culture supernatant. The NH2-terminal amino acid sequence, the COOH-terminal amino acid (Ala), and the amino acid composition of the human NGF were identical to those deduced from the nucleotide sequence of the human NGF gene. The recombinant human NGF was composed of 120 amino acid residues. Three disulfide linkages were determined to be Cys15-Cys80, Cys-58-Cys108, and Cys68-Cys110; the locations were identical to those in the mouse 2.5S NGF molecule. The specific biological activity of the recombinant human NGF was comparable with that of authentic mouse 2.5S NGF as determined by stimulation of neurite outgrowth from PC12 cells.     

Human acid sphingomyelinase.

Human acid sphingomyelinase (haSMase, EC 3.1.4.12) catalyzes the lysosomal degradation of sphingomyelin to ceramide and phosphorylcholine. An inherited haSMase deficiency leads to Niemann-Pick disease, a severe sphingolipid storage disorder. The enzyme was purified and cloned over 10 years ago. Since then, only a few structural properties of haSMase have been elucidated. For understanding of its complex functions including its role in certain signaling and apoptosis events, complete structural information about the enzyme is necessary. Here, the identification of the disulfide bond pattern of haSMase is reported for the first time. Functional recombinant enzyme expressed in SF21 cells using the baculovirus expression system was purified and digested by trypsin. MALDI-MS analysis of the resulting peptides revealed the four disulfide bonds Cys120-Cys131, Cys385-Cys431, Cys584-Cys588 and Cys594-Cys607. Two additional disulfide bonds (Cys221-Cys226 and Cys227-Cys250) which were not directly accessible by tryptic cleavage, were identified by a combination of a method of partial reduction and MALDI-PSD analysis. In the sphingolipid activator protein (SAP)-homologous N-terminal domain of haSMase, one disulfide bond was assigned as Cys120-Cys131. The existence of two additional disulfide bridges in this region was proved, as was expected for the known disulfide bond pattern of SAP-type domains. These results support the hypothesis that haSMase possesses an intramolecular SAP-type activator domain as predicted by sequence comparison [Ponting, C.P. (1994) Protein Sci., 3, 359-361]. An additional analysis of haSMase isolated from human placenta shows that the recombinant and the native human protein possess an identical disulfide structure.