The disulphide bridges of a mouse immunoglobulin G1 protein

[(35)S]Cystine-labelled immunoglobulin MOPC21 (IgG1) was prepared from myeloma cells in tissue culture. Carrier myeloma protein was added and the protein was digested with pepsin. The digest was fractionated on Sephadex G-50 into two fractions, further digested with trypsin and again fractionated on Sephadex. Disulphide-bridge peptides were purified by electrophoresis and chromatography and identified by radioautography. A peptide of 96 residues was isolated, which contains both the heavy-light interchain disulphide bridge and all the inter-heavy-chain disulphide bridges. Other peptides were isolated, accounting for all the intrachain disulphide bridges (which could be placed by homology with proteins of other species), except for the variable section of the light chain. Sequences describing this missing disulphide bridge were obtained from totally reduced and alkylated light chains. Peptides related to the interchain disulphide-bridge peptide were isolated from partially reduced and alkylated myeloma protein and from totally reduced heavy chain. The interchain disulphide-bridge peptide was placed at the C-terminal position of the F(ab')(2) fragment, prepared by digestion of the protein with pepsin at pH4.0. Sequences from the heavy-chain intrachain disulphide bridges of MOPC 21 immunoglobulin are compared with homologous sequences from mouse myeloma proteins of other subclasses and proteins of other species.     

Fc and Fab Fragments from IgG<Subscript>2</Subscript> Human Immunoglobulins Characterized

Papain digestion of 7S immunoglobulin G (IgG) produces two 3.5S Fab fragments and one 3.5S Fc fragment^1–8. The Fab fragment contains one light chain and one Fd fragment and is still able to combine specifically univalently with antigen. The Fc fragment is a dimer of the carboxyl terminal half of the heavy chain. Pepsin splits 7S IgG into some small peptides derived from Fc and one 5S F(ab′)₂ fragment, which contains both antigen-binding sites. Based on this information, some investigators^6,7 have postulated that pepsin splits the γ chains at the C-terminal side of the inter-heavy chain disulphide bridges, whereas papain splits at the N-terminal side of the inter-heavy chain disulphide bridges. We report here evidence that this model does not apply to all IgG subclasses. In the case of human IgG₂ subclass myeloma proteins, papain splits initially at the C-terminal side of inter-heavy chain disulphide bridges. We also show that the amino-acid sequence of the Fc fragment of human IgG₂ subclass so far determined has approximately 95% homology with that of human IgG₁ and IgG₄ subclasses reported by others^9–15.     

The disulphide bridges of immunoglobulin κ-chains

The arrangement of the disulphide bridges of the major component of the light chains of immunoglobulins (kappa-chains) has been studied in the Bence-Jones proteins. Three disulphide bridges have been found. An interchain bridge at the C-terminus has been shown to occur in the dimers of all the proteins studied and was characterized by symmetrical peptides. In the monomer form, the C-terminal half-cystine of the corresponding peptides was linked to a lone half-cystine residue. A second common disulphide-bridge peptide in which a single amino acid difference could be related to the Inv factors of the individual proteins was found in Bence-Jones proteins and in the kappa-chains of normal and abnormal immunoglobulins. Peptides characteristic of a third disulphide bridge studied in three specimens were found to have differences in some residues, but also striking similarities. A methionine peptide has also been characterized in two specimens as a by-product of the technique employed. It is suggested that a general manner of folding may be a common feature of the heterogeneous population of kappa-chains: one bridge which folds an invariable stretch of the chain, another bridge which folds a stretch that varies from protein to protein, and a bridge at the C-terminus which is the interchain link.     

The disulphide bonds of an Asian influenza virus neuraminidase

The arrangement of the disulphide bonds in the pronase-released neuraminidase heads of the Asian influenza virus A/Tokyo/3/67 have been examined by cyanogen bromide fragmentation, enzymic digestion and diagonal peptide mapping. There are 9 intrachain disulphide bridges and one interchain bridge which links pairs of monomers at the distal end of the stalk region of the neuraminidase tetramer. The disulphide bond arrangements of the remaining 3 half-cystine residues in the membrane-embedded stalk region of the neuraminidase were not examined.     

Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases

Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish the chemistry required for the generation of a split-synthesis library, two substrates containing an interchain disulphide bond, a fluoroescent probe and a quencher were synthesized. The library consists of a Cys residue flanked by randomized amino acid residues at both sides and the fluoroescent Abz group at the amino terminal. All the 20 natural amino acids except Cys were employed. The library was linked to PEGA‒beads via methionine so that the peptides could be selectively removed from the resin by cleavage with CNBr. A disulphide bridge was formed between the bead‒linked library and a peptide containing the quenching chromophore (Tyr(NO₂)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluoroescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were also synthesized. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Studies on alkaline phosphatase. Transientstate and steady-state kinetics of Escherichia coli alkaline phosphatase

1. Reduction of a 19s immunoglobulin M with 3mm-mercaptoethanol or 0.05-0.5mm-dithiothreitol followed by alkylation gave sedimentation patterns indicating products compatible with structures consisting of one, two, three, four and five 7s sub-units. This supports the concept of a five-sub-unit structure for immunoglobulin M. 2. Reduction with 0.125mm-dithiothreitol or 20mm-cysteine produced 7s sub-units that could not be dissociated into chains in m-propionic acid. 3. By labelling (with iodo[2-(14)C]acetic acid) the thiol groups liberated during reduction with 0.125mm-dithiothreitol, it was possible to identify the tryptic peptides involved in the disulphide bridges that link the 7s sub-units together (inter-sub-unit bridges). 4. By further reducing and labelling (with iodo[2-(14)C]acetic acid) the 7s sub-units produced by 0.125mm-dithiothreitol, it was possible to identify tryptic peptides derived from intra-sub-unit bridges. 5. Sub-units produced by reduction with 20mm-cysteine proved to be unsuitable for distinguishing between inter-sub-unit bridges and intra-sub-unit bridges. 6. The possible arrangement of the interchain disulphide bridges was deduced.     

Disulphide bridges of the heavy chain of human immunoglobulin G2

Amino acid sequences around the disulphide bridges of the heavy chain of an immunoglobulin of the gamma2 subclass have been studied. The protein was digested with pepsin and the digest fractionated by Sephadex. Screening of the eluate by one-dimensional electrophoresis of oxidized and unoxidized samples was used as an assay and pools of fractions were prepared. Identification by diagonal electrophoresis of several inter- and intra-chain disulphide bridges was done on the pooled fractions. The inter-heavy-chain bridged peptide included four cystine residues. Comparison with proteins of other human subclasses indicated that the intrachain bridges identified are the bridges of the invariable section of gamma2 heavy chains. The amino acid sequence of one cysteic acid peptide that may have been derived from the variable part of the molecule was determined. Partial reduction followed by carboxymethylation with radioactive iodoacetate of two proteins of the gamma2 class showed a number of labelled peptides that could be identified as being related to the inter-chain bonded cystine residues.     

Immunochemical characterization of human plasma fibronectin.

Human plasma fibronectin has been purified by a non-denaturing affinity chromatography procedure [Vuento & Vaheri, (1979) Biochem.J. 183, 331--337], and antisera have been raised by immunizing rabbits with the native protein. The antisera reacted strongly with native fibronectin, but only weakly with reduced and alkylated fibronectin or with heat-denaturated fibronectin. Denaturation also affected the haemagglutinating and gelatin-binding activities of fibronectin and increased its susceptibility to proteolytic degradation. The antisera reacted with fragments of fibronectin obtained by proteolysis with plasmin. Large fragments (mol.wt. 180000--200000), lacking the region harbouring the interchain disulphide bridges but containing the sites responsible for gelatin-binding and haemagglutinating activity, showed as intense a reaction with the antisera as intact fibronectin. Smaller peptides showed a weaker reaction. All fragments tested showed sensitivity to denaturation in their reaction with the antisera. The results were interpreted as showing that: (1) native fibronectin has an ordered conformation that is easily perturbed by denaturation; (2) most of the antigenic determinants of the protein are dependent on conformation; (3) the region of the fibronectin molecule containing the interchain disulphide bridges has only few antigenic determinants; and (4) covalent interaction of the two subunits does not contribute to the antigenic structure recognized by rabbit antisera. The observed correlation between the antigenic activity and a structural and functional intactness of fibronectin suggests that the antibodies to native fibronectin could be used as a conformational probe in studies on this protein.     

Stepwise cleavage of rabbit immunoglobin G by papain and isolation of four types of biologically active Fc fragments

Four types of Fc fragments of different sizes were isolated by papain treatment of rabbit immunoglobulin G under various conditions and by subsequent chromatographic procedures. 1. Brief digestion at neutral pH without reduction produced a molecule in which the Fab and Fc fragments were still linked by a pair of labile disulphide bridges, and the Fc fragment released by cleaving these bonds, called 1Fc fragment, contained a portion of the ;hinge' region including an interchain disulphide bridge. Both complement-binding and guinea-pig skin-binding activities were retained by this fragment, which had mol. wt. 48000. 2. Prolonged digestion at neutral pH of immunoglobulin G whose labile inter-heavy-chain disulphide bridges had been reduced removed the ;hinge' region, giving mFc fragments (mol. wt. 46000), which lacked the capacity to bind guinea-pig skin but retained the antigenic as well as the complement-binding activities of 1Fc fragment completely. 3. Digestion at pH5.0 yielded a smaller fragment, sFc (mol. wt. 40000), which was no longer able to bind complement. Though the antigenic structure was intact, sFc fragment was curiously unable to precipitate with antibodies to the N-terminal determinants. 4. Fragment stFc (mol. wt. 25000), representing the C-terminal portion of Fc fragment, was formed from all the larger fragments by digestion at pH4.5. Only the C-terminal antigenic determinants were retained by stFc fragment.     

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.     

The incorporation of J chain into reassembled human immunoglobulin M

Human IgM (immunoglobulin M) was reduced with 24mm-mercaptoethylamine. This atreatment resulted in complete dissociation to IgMs subunits and free J chain. Intr-subunit interchain disulphide bonds remained intact. The mixture then was encouraged to reoxidize. The schlieren pattern of the reoxidized mixture showed the presence of a considerable quantity of IgM in addition to residual IgMs. The isolated reassembled IgM did not dissociate in 5m-guanidinium hydrochloride. It apparently contained the same amount of covalently attached J chain as did native IgM. The J chain was a part of the high-molecular-weight Fc fragment obtained from the reassembled IgM.     

NIH-3T3 cells transformed with a ras oncogene exhibit a protein kinase C-mediated inhibition of agonist-stimulated Ca2+ inflow.

Bacillus thuringiensis produces a 130-140 kDa insecticidal protein in the form of a bipyramidal crystal. The protein in the crystals from the subspecies kurstaki HD-1 and entomocidus was found to contain 16-18 cysteine residues per molecule, present primarily in the disulphide form as cystine. Evidence that all the cysteine residues form symmetrical interchain disulphide linkages in the protein crystal was obtained from the following results: (i) the disulphide diagonal procedure [Brown & Hartley (1966) Biochem. J. 101, 214-228] gave only unpaired cysteic acid peptides in diagonal maps; (ii) the disulphide bridges were shown to be labile in dilute alkali and the crystal protein could be released quantitatively with 1 mM-2-mercaptoethanol; (iii) the thiol groups of the released crystal protein were shown by competitive labelling [Kaplan, Stevenson & Hartley (1971) Biochem. J. 124, 289-299] to have the same chemical properties as exposed groups on the surface of the protein; (iv) the thiol groups in the released crystal protein reacted quantitatively with iodoacetate or iodoacetamide. The finding that all the disulphide linkages in the protein crystal are interchain and symmetrical accounts for its alkali-lability and for the high degree of conservation in the primary structure of the cystine-containing regions of the protein from various subspecies.     

Primary structure of the B-chain of human plasmin.

The primary structure of the human plasmin B-chain has been determined. It consists of 230 residues divided in three cyanogen bromide fragments: The amino-terminal 24 residues, the carboxy-terminal three residues and the middle 203 residues. Sequence detemination was performed on the tryptic and the chymotryptic peptides obtained from the main cyanogen bromide fragment of this chain. Owing to similarities between some of the overlapping chymotryptic peptides, two different sequences were possible from these results. However, since the homologies with the pancreatic serine proteases and also the B-chains of thrombin and factor XA are pronounced, the arrangement still could be settled. By peptic digestion of partially reduced and S-carboxymethylated B-chain it was shown that there are two interchain disulphide bridges, which connect the A and B-chains of plasmin, involving Cys-5 and Cys-105 from the B-chain. The intrachain disulphides in the B-chain seem to be situated exactly as in chymotrypsin as partly judged from homologies.     

Localization of carbohydrate attachment sites and disulfide bridges in limulus alpha 2-macroglobulin. Evidence for two forms differing primarily in their bait region sequences

The primary structure determination of the dimeric invertebrate alpha(2)-macroglobulin (alpha(2)M) from Limulus polyphemus has been completed by determining its sites of glycosylation and disulfide bridge pattern. Of seven potential glycosylation sites for N-linked glycosylation, six (Asn(275), Asn(307), Asn(866), Asn(896), Asn(1089), and Asn(1145)) carry common glucosamine-based carbohydrates groups, whereas one (Asn(80)) carries a carbohydrate chain containing both glucosamine and galactosamine. Nine disulfide bridges, which are homologues with bridges in human alpha(2)M, have been identified (Cys(228)-Cys(269), Cys(456)-Cys(580), Cys(612)-Cys(799), Cys(657)-Cys(707), Cys(849)-Cys(876), Cys(874)-Cys(910), Cys(946)-Cys(1328), Cys(1104)-Cys(1155), and Cys(1362)-Cys(1475)). In addition to these bridges, Limulus alpha(2)M contains three unique bridges that connect Cys(361) and Cys(382), Cys(1370) and Cys(1374), respectively, and Cys(719) in one subunit with the same residue in the other subunit of the dimer. The latter bridge forms the only interchain disulfide bridge in Limulus alpha(2)M. The location of this bridge within the bait region is discussed and compared with other alpha-macroglobulins. Several peptides identified in the course of determining the disulfide bridge pattern provided evidence for the existence of two forms of Limulus alpha(2)M. The two forms have a high degree of sequence identity, but they differ extensively in large parts of their bait regions suggesting that they have different inhibitory spectra. The two forms (Limulus alpha(2)M-1 and -2) are most likely present in an approximately 2:1 ratio in the hemolymph of each animal, and they can be partially separated on a Mono Q column at pH 7.4 by applying a shallow gradient of NaCl.     

Alternate Positions for the Sulphydryl Group in β-Lactoglobulin: the Significance for Sulphydryl Location in Other Proteins

EARLIER studies of the location of the single cysteine residue and the two disulphide bridges in bovine β-lactoglobulins A and B¹, for each of which the monomer is a single chain of 162 residues and 18,000 molecular weight^2,3, led to the conclusion that the sulphydryl group is at position 69 and that the disulphides bridge positions 123 to 160 and 57 to 70. These results were based on diagonal peptide studies⁴ and on the composition of peptides in which the sulphydryl group had been labelled with ¹⁴C-iodoacetamide, the disulphide bridges being left intact. Use was made of the partial amino-acid sequence given by Frank and Braunitzer⁵ and the reasonable assumption was made that the sulphydryl occurred in only one position. Subsequently, Shaw⁶ has shown that the sequence of Frank and Braunitzer⁵ showing Cys residues adjacent at positions 69 and 70 is incorrect and that they are separated by a glutamine, the sequence for positions 67 to 71 for the Bvariant being Ala.Cys.Gln.Cys.Leu. Autoradiography of the dansyl amino-acid derivatives formed during the sequence determination of this pentapeptide indicated that both residues 68 and 70 seemed to have been labelled and so we have given further consideration to the sulphydryl location. It has been found that although it does occur at 68, with 57 and 70 disulphide bridged, there is also an equal amount of protein present with the sulphydryl at 70, with 57 and 68 disulphide bridged. We discuss this additional finding here and the significance for the determination of the location of sulphydryl groups in other proteins.     

The disulphide bridges and soluble tryptic peptides of calf rennin

1. Cysteic acid peptides from various digests of calf rennin were purified by diagonal paper electrophoresis. 2. The amino acid sequence of these peptides accounts for 38 amino acids around three unique disulphide bridges in rennin. 3. One bridge connects two acidic regions of the chain, one forms a loop of five residues and the other a loop of six residues. 4. These bridges are homologous with those of hog pepsin. 5. Tryptic peptides from the C-terminus of rennin account for 22 residues, 17 of which are homologous with the C-terminus of pepsin. 6. Altogether, sequences accounting for 94 of the 270 residues in rennin are described and the degree of homology with pepsin approximates to 70%.     

Isolation and biochemical characterization of the alpha- and beta-subunits of glycoprotein IIb of human platelet plasma membrane.

The alpha- and beta-subunits of glycoprotein IIb (GPIIb) of human platelet plasma membrane were isolated in fully reduced, partially reduced and alkylated, and fully alkylated forms, by size-exclusion chromatography after reduction of pure GPIIb. The sugar moiety of GPIIb alpha accounts for 16.4% of its total weight, whereas that of GPIIb beta accounts for only 10.2%. The molar percentages (per 100 mol of total amino acids) of neuraminic acid and galactose in the alpha-subunit more than double those in the beta-subunit, whereas galactosamine is present only in GPIIb alpha. From the amino acid and sugar compositions the acidic nature of both subunits was confirmed. The Mr values obtained, 114,000 for GPIIb alpha and 22,200 for GPIIb beta, are in very good agreement with those obtained by physical methods. We found by stepwise reduction of pure GPIIb with dithioerythritol that GPIIb alpha and GPIIb beta are joined by a single interchain disulphide bridge, while the remaining half-cystine residues participate in intrachain bonds, six in GPIIb alpha and one in GPIIb beta, the intersubunit disulphide bond being that reduced first. Neither of the two subunits is liberated from isolated plasma membranes when this GPIIb interchain bond is reduced in isolated membranes.     

The disulphide bridges in a cellobiohydrolase and an endoglucanase from Trichoderma reesei.

The positions of the disulphide bridges of the 1,4-beta-glucan cellobiohydrolase (CBH I) of the fungus Trichoderma reesei have been investigated. The results can be summarized as follows. (1) The enzyme contains 12 disulphide bridges and no free cysteine residues. (2) The location of six disulphide bridges have been determined experimentally. (3) The bonding patterns of the two disulphide bridges in the C-terminal region is suggested on the basis of internal homology. (4) The remaining four disulphide bridges are put into two groups, each containing four half-cystine residues where two are adjacent. (5) A repeating bonding pattern is observed along the peptide chain and a non-local disulphide bond with an unusually long separation distance links the N-terminal and the C-terminal region. (6) The disulphide-bonded CNBr peptides of a 1,4-beta-glucan glucanohydrolase (endoglucanase II) from T. reesei have been isolated and a disulphide bonding pattern is suggested on the basis of the sequence homology between the two enzymes.     

Disulphide bridges of bovine factor X.

Evidence is presented for the disulphide bridges in bovine Factor X. The protein was degraded by chemical and enzymic means, and all 12 disulphide bridges were isolated in separate peptides except for bridges nos. 6/7 in the light chain. All the disulphide bridges were found to be in positions corresponding to those found in other homologous domains. This report is the first verification of an epidermal-growth-factor-homologous domain having the same disulphide-bonding pattern as that found in mouse epidermal growth factor.     

A new method for the selective isolation of cysteine-containing peptides. Specific labelling of the thiol group with a hydrophobic chromophore.

A new method for the selective isolation of cysteine-containing peptides was designed. The method is based on the specific labelling of thiol groups with a hydrophobic chromophore followed by enzymic fragmentation of the labelled protein and reversed-phase high-pressure liquid-chromatographic separation of the peptide mixture. This new method has several distinct advantages: (1) the hydrophobic-chromophore-labelled cysteine-containing peptides are easily separated from non-cysteine-containing peptides by reversed-phase high-pressure liquid chromatography; (2) only cysteine-containing peptides are detected in the visible region with sensitivity at the low picomole level; this high sensitivity allows isolation of nanogram amounts of pure cysteine-containing peptide; (3) during sequence determination of the chromophore-labelled cysteine-containing peptides, the cysteine residues are released as coloured anilinothiazolinone derivatives and can be detected directly in the picomole range; (4) with proteins bearing several disulphide groups, each disulphide group may undergo a different degree of reduction, and therefore the recovery of individual cysteine-containing peptides may be used to deduce the disulphide linkages present in the native protein. Two thiol-specific reagents, 4-dimethylaminoazobenzene-4'-iodoacetamide and 4-dimethylaminoazobenzene-4'-N-maleimide, were synthesized and characterized. The method was successfully used to isolate five cysteine-containing peptides from a completely reduced monoclonal-antibody kappa-light chain raised against the azobenzenearsonate determinant and six cysteine-containing peptides from a kappa-light chain raised against streptococcal group A polysaccharide. The principle of this method is applicable to the isolation of any peptide containing amino acid residues that can be specifically labelled with a hydrophobic chromophore.