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.     

Complete localization of the intrachain disulphide bonds and the N-glycosylation points in the alpha-subunit of human platelet glycoprotein IIb.

Glycoprotein IIb (GPIIb), one of the two molecular components of the inducible receptor for fibrinogen on the platelet surface, is formed from two subunits, GPIIb alpha (114 kDa) and GPIIb beta (22.5 kDa), joined by a single disulphide bond. CNBr cleavage of GPIIb, together with tryptic or endoproteinase Lys-C digestion of some of the isolated CNBr peptides, followed by amino acid and N-terminal sequence analysis of the isolated fragments, allowed us to locate unambiguously all the unknown disulphide bonds and the N-glycosylation points in platelet GPIIb. It could be established that each cysteine residue in GPIIb, beginning at alpha-Cys-56, is disulphide-bonded to its nearest neighbour in the amino acid sequence. Given the extensive structural similarity among the two-chain alpha-subunits of Arg-Gly-Asp adhesion receptors and the conservative positions of cysteine residues in their amino acid sequences, the intrachain and interchain disulphide-bond pattern found here in GPIIb will most probably be conserved in all two-chain alpha-subunits of these receptors. The N-linked glycosylation points found here in platelet GPIIb are the same as the five N-glycosylated asparagine residues suggested after cDNA sequencing of human erythroleukaemic-cell GPIIb [Poncz, Eisman, Heindenreich, Silver, Vilaire, Surrey, Schwartz & Bennett (1987) J. Biol. Chem. 262, 8476-8482]. Some of the general features of the structure of GPIIb, such as the existence of distinct domains in the alpha- and beta-subunits, as well as the identification of well-defined points in its external topography, are discussed.     

Primary structure of human alpha 2-macroglobulin. V. The complete structure

The primary structure of the tetrameric plasma glycoprotein human alpha 2-macroglobulin has been determined. The identical subunits contain 1451 amino acid residues. Glucosamine-based oligosaccharide groups are attached to asparagine residues 32, 47, 224, 373, 387, 846, 968, and 1401. Eleven intrachain disulfide bridges have been placed (Cys25-Cys63, Cys228-Cys276, Cys246-Cys264, Cys255-Cys408, Cys572-Cys748, Cys619-Cys666, Cys798-Cys826, Cys824-Cys860, Cys898-Cys1298, Cys1056-Cys1104, and Cys1329-Cys1444). Cys-447 probably forms an interchain bridge with Cys-447 from another subunit. The beta-SH group of Cys-949 is thiol esterified to the gamma-carbonyl group of Glx-952, thus forming an activatable reactive site which can mediate covalent binding of nucleophiles. A putative transglutaminase cross-linking site is constituted by Gln-670 and Gln-671. The primary sites of proteolytic cleavage in the activation cleavage area (the "bait" region) are located in the sequence: -Arg681-Val-Gly-Phe-Tyr-Glu-. The molecular weight of the unmodified alpha 2-macroglobulin subunit is 160,837 and approximately 179,000, including the carbohydrate groups. The presence of possible internal homologies within the alpha 2-macroglobulin subunit is discussed. A comparison of stretches of sequences from alpha 2-macroglobulin with partial sequence data for complement components C3 and C4 indicates that these proteins are evolutionary related. The properties of alpha 2-macroglobulin are discussed within the context of proteolytically regulated systems with particular reference to the complement components C3 and C4.     

The positions of the disulfide bonds and the glycosylation site in a lectin of the acorn barnacle Megabalanus rosa

The positions of the interchain and intrachain disulfide bonds and the glycosylation site in a lectin of the acorn barnacle Megabalanus rosa were determined. The lectin (Mr 140,000) is composed of the same subunit (Mr 22,000) which is cross-linked by disulfide bonds to form a dimer. Intact lectin yielded two fragments, CB1 and CB2, by cleavage with cyanogen bromide. One intrachain and two interchain disulfide bonds were identified as Cys-53-Cys-61, Cys-14-Cys-50' and Cys-50-Cys-14', respectively, by enzymatic digestion and Edman degradation of CB1. Two intrachain disulfide bonds were determined as Cys-78-Cys-168 and Cys-144-Cys-160 by enzymatic digestion of CB2. The two intrachain disulfide bonds are well conserved through all invertebrate lectins and calcium-dependent animal lectins. S-Carboxamidomethylated lectin was digested with Staphylococcus aureus V8 proteinase and separated by reversed-phase HPLC. Glycopeptides were detected by the 4-N,N-dimethylamino-4'-azobenzene sulfonyl hyrazide method. Sequence analyses of the glycopeptides showed that a carbohydrate chain attached to Asn-39.     

Agonist-specific structural rearrangements of integrin alpha IIbbeta 3. Confirmation of the bent conformation in platelets at rest and after activation

Concrete structural features of integrin alpha(IIb)beta(3) on the surface of platelets (at rest and after activation) have been obtained from epitope maps based on cross-competition among monoclonal antibodies directed against the alpha(IIb) subunit calf-2 domain and the beta(3) subunit betaA domain of alpha(IIb)beta(3). At rest, the observed intersubunit interface is formed by the sequence stretches beta(3)-(150-216), alpha(IIb) light chain-(1-92), and alpha(IIb) heavy chain-(826-856); and the alpha(IIb) interchain interface is formed by the two latter sequence stretches, disulfide-bonded between alpha(IIb) heavy chain Cys(826) and alpha(IIb) light chain Cys(9). These structural features agree with those observed in the alpha(IIb)beta(3) rudimentary connectivity map in solution and with the alpha(v)beta(3) V-shaped crystal structure (Xiong, J.-P., Zhang, R., Dunker, R., Scott, D. L., Joachimiak, A., Goodman, S. L., and Arnaout, M. A. (2001) Science 294, 339-345), but they disagree with the domain disposition suggested by the actual ultrastructural model. The epitope maps in platelets activated by ADP, thrombin receptor activation peptide, and arachidonic acid differ not only from those in platelets at rest, but also among themselves. The structural rearrangements observed confirm the presence in activated platelets of the crystallographically observed knee and argue against the switchblade mechanism proposed for activation (Beglova, N., Blacklow, S. C., Takagi, J., and Springer, T. A. (2002) Nat. Struct. Biol. 9, 282-287), demonstrate the existence of alpha(IIb)beta(3) agonist-specific activation states, explain the specificity for ligand binding and functional inhibition for some agonists, and predict the existence of agonist-specific final effectors and receptor activation mechanisms. The distinct non-reciprocal competition patterns observed at rest and after activation support the agonist-specific activation states and the existence of intrasubunit and intersubunit allosteric effects, previously proposed as the mechanism for alpha(IIb)beta(3) transmembrane activation.     

Chemical structure of two fragments of human serum albumin and their location in the albumin molecule.

1. 'Inhibitor fragment' isolated from human serum albumin degraded by rabbit cathepsin D is composed of one peptide chain with two intrachain disulphide bonds. There are two kinds of inhibitor molecules having different N-terminal amino acids: one is threonine and the other glutamine. 2. Fragment F1, isolated from inhibitor degraded by trypsin, is composed of two chains linked by a disulphide bond. There are three kinds of fragment F1. All have one alpha chain in common, which has an intrachain disulphide bond. They differ by the nature of the chain, which is linked to the alpha chain by a disulphide bond. The epsilon chain is present in trace amounts. The two other chains, beta and gamma, differ by their C-terminal amino acid, which is respectively arginine and lysine. 3. Inhibitor is composed of the last 92 or 89 residues of the human albumin molecule and fragment F1 is composed of two parts of this C-terminal portion of the albumin molecule.     

Detection of disulphide bonds and localization of interchain linkages in the third (C3) and the fourth (C4) components of human complement.

Disulphide bonds contribute significantly to the maintenance of structural/functional integrity of many proteins. Therefore it was of interest to study the distribution and the effect of disulphides on conformation of complement components C3 and C4. These proteins are precursors of several fragments with various binding sites and distinct physiological functions. The constituents of C3c (beta, alpha 27, alpha 43) and those of C4c (beta, alpha 27, alpha 16, gamma) were investigated, since other fragments of C3 or C4 do not participate in interchain linkages. Inter-and intra-chain disulphide bonds in C3c and C4c were localized by using a modification of conventional SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis such that the change in mobility of disulphide-bond-containing proteins can be detected throughout the transition from a non-reduced to a fully reduced state. Several forms of the alpha 43 fragment from C3, and of the gamma-chain of C4, with different mobilities can exist, depending on the number of intra-chain disulphide bonds reduced. The intermediates (heterodimers) generated by a partial reduction of C3c or C4c were characterized by two-dimensional SDS/polyacrylamide-gel electrophoresis performed in the absence, then in the presence, of beta-mercaptoethanol. The inter-chain linkages in C3c were determined to be beta-alpha 27 and alpha 27- alpha 43, thus indicating the presence of only one interchain bond in C3. The two interchain bonds in C4c are beta-alpha 27 and alpha 16-gamma. The third interchain bond in C4 (alpha 27-gamma, tentative) remains to be determined.     

Comparative study of the glycosylation of platelet glycoprotein GPIIb/IIIa and the vitronectin receptor. Differential processing of their beta-subunit.

The platelet glycoprotein GPIIb/IIIa and the vitronectin receptor (VNR) are alpha beta-heterodimeric proteins and share the same beta-subunit. By performing swainsonine treatment and digestion with endoglycosidase H (Endo H), we showed that the heavy chains of GPIIb and VNR alpha are glycosylated by complex-type oligosaccharide chains, and provided the first evidence for the presence of one complex carbohydrate residue on their light chains. The proteolytic cleavage of pro-GPIIb and the acquisition of Endo H-resistance are independent events occurring in the same Golgi compartment. We demonstrated the Endo H-sensitivity of GPIIIa and VNR beta in all cellular systems tested. In addition, this beta-subunit is differently glycosylated according to whether it is associated with GPIIb or VNR alpha, one carbohydrate chain being processed to the complex type on GPIIIa, but not on VNR beta.     

Substructural analysis of the insulin receptor by microsequence analyses of limited tryptic fragments isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence or presence of dithiothreitol

Human placental insulin receptor contains 47 Cys per an alpha beta dimer. Most of the 94 Cys in an intact alpha 2 beta 2 receptor are expected to form interchain or intrachain disulfide bonds, since there appears to be only one free cysteine residue in each beta subunit. In order to gain more insight into the three-dimensional organization of the insulin receptor, we have used limited trypsin digestion, SDS-PAGE, and protein microsequencing. The present study revealed the following; major tryptic cleavages occurred at alpha 164, alpha 270, alpha 582, and beta 1115, generating Mr 175,000, 130,000, 100,000, 70,000, and 55,000 disulfide-linked complexes. Under reducing conditions, tryptic fragments of Mr values = 30,000, 70,000, 20,000, 55,000, and 20,000 were identified to be alpha(1-164), alpha(165-582), alpha(165-270), alpha(271-582), and alpha(583-C-terminal), respectively. The major beta subunit tryptic fragment of Mr = 55,000 was assumed to have beta(724-1115) or beta(N-terminal-392). The Mr 175,000 complex appeared to contain two alpha(1-164) and two alpha(165-582), whereas the Mr 70,000 complex contained alpha(583-C-terminal) and beta(724-1115). Tryptic cleavage at alpha 582 apparently produced one Mr 175,000 and two Mr 70,000 complexes, suggesting that the alpha(583-C-terminal) domain interacts with the extracellular domain of the beta subunit by disulfide bonds. Tryptic cleavage at alpha 270 resulting in a formation of one Mr 100,000 complex consisting of two alpha(1-270) and two Mr 130,000 complexes consisting of alpha(271-C-terminal) and beta(724-1115) suggests that Cys residues involved with disulfide bonds between the two alpha subunits are located in the alpha(1-270) domain. The identification of the Mr 55,000 complex consisting of small tryptic fragments between alpha(122-270) indicates that 40 Cys residues in the two alpha(122-270) domains are inter- and intramolecularly associated by disulfide bonds. The alpha(1-121) domain does not appear to be linked to any other domains by disulfide bonds. These results are consistent with the structural model that the N-terminal domains of alpha subunits (122-270) are disulfide-linked together while the C-terminal domain (583-C-terminal) of the alpha subunit is linked to the N-terminal domain of the beta subunit by disulfide bonds.     

Role of the transmembrane and cytoplasmic domains in the assembly and surface exposure of the platelet integrin GPIIb/IIIa.

Integrins are alpha beta heterodimers that play a major role in cell-cell contacts and in interactions between cells and extracellular matrices. Identification of structural domains that are critical for the expression of such receptors at the cell surface in a functional conformation is one of the major issues that has not yet been resolved. In the present study, the role of the cytoplasmic and transmembrane domains of each of the subunits has been examined using platelet GPIIb/IIIa as a prototypic integrin. GPIIb/IIIa (alpha IIb/beta 3) is a member of the integrin family and functions as a receptor for fibrinogen, fibronectin, von Willebrand factor, and vitronectin at the surface of activated platelets. Human megakaryocyte GPIIb and GPIIIa cDNAs were used to create a GPIIb mutant coding for the extracellular GPIIb heavy chain alone (GPIIb delta 1) and a GPIIIa mutant lacking the transmembrane and cytoplasmic domains (GPIIIa delta m). Full length and mutant cDNAs were subcloned into the expression vector pECE and used to transfect COS cells. The formation of heterodimers and their cellular localization was analyzed by immunoprecipitation and immunofluorescence labeling using anti-platelet GPIIb/IIIa antibodies. We show here that the extracellular domains of alpha and beta subunits are able to form a heterodimer, although with a lower efficiency, in the absence of the transmembrane and cytoplasmic domains. The presence of the cytoplasmic and transmembrane domains in the alpha subunit is, however, necessary for expression at the surface of the cell whereas the corresponding domains of the beta subunit are not required.     

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.     

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.     

Co-binding studies on Hb M Iwate. Allostery of a T state haemoglobin.

The mutant haemoglobin Hb M Iwate alpha 2Mmet87His leads to Tyr beta 2, is characterized by a stable T structure and a low ligand affinity. Sigmoidal CO-binding isotherms of symmetrical shape with Hill coefficients of n = 1.4 at pH 6 to n = 1.9 at pH 10 and the differences in the mean affinity (PCO(1/2)) and the affinity of the first ligand-binding beta subunit (1/L1 greater than Pco(1/2)) are the evidence for the cooperativity. The comparison of the Bohr effects of the two valency hybrid states (alpha 2Mmet beta met beta deoxy alpha 2Mmet beta 2deoxy) in the absence of and in the presence of polyphosphates leads to an indirect proof of pH-dependent subunit-subunit interaction. Inositol hexaphosphate-binding suppresses cooperativity in the pH range 5.5-8 (n = 1). Above pH 8 hte cooperativity increases to a final value of n = 1.9 at pH greater than 10, which is identical to that of stripped Hb M Iwate. The CO binding to the first binding site exhibits a Bohr effect. Polyphosphate anions have no influence on the CO binding of the first binding site. The heterotropic effects are discussed as intrachain effects (Bohr effect of the first binding site) and interchain effects (Bohr effect of Pco(1/2); influence of polyphosphates).     

The epsilon subunit and inhibitory monoclonal antibodies interact with the carboxyl-terminal region of the beta subunit of Escherichia coli F1-ATPase

The epitopes of two classes of monoclonal antibody and the binding site for the epsilon subunit have been mapped to the carboxyl-terminal region of the beta subunit of Escherichia coli F1-ATPase using partial CNBr cleavage, weak acid hydrolysis, and Western blots. One class of antibody, B-I, inhibits ATPase activity; the other class, B-II, recognizes an epitope not exposed on the surface of intact F1. Data from two-dimensional gels and blots of beta cleaved with CNBr/weak acid showed that the B-I epitope lies between Asp-381 and the carboxyl-terminal Leu-459, and the B-II epitope lies between Asp-345 and Met-380. Weak acid hydrolysis of the beta-epsilon product obtained by cross-linking F1 with a water-soluble carbodiimide yielded a fragment containing epsilon and a 13-kDa carboxyl-terminal fragment of beta indicating that epsilon interacts with this portion of beta as well. Fab fragments from the B-I antibody beta-6 could be cross-linked to the epsilon subunit in native F1 by various cross-linking agents demonstrating that the antibody and the epsilon subunit occupy adjacent, nonoverlapping sites on the beta subunit. Implications of these results for the roles of the epsilon subunit and of the carboxyl-terminal region of the beta subunit in F1 are discussed.     

Nonclottable fibrin obtained from partially reduced fibrinogen: characterization and tissue plasminogen activator stimulation.

Out of 29 disulfide bonds in human fibrinogen, 7 were cleaved during limited reduction under nondenaturing conditions in calcium-free buffer: 2 A alpha 442Cys-A alpha 472Cys and 2 gamma 326Cys-gamma 339Cys intrachain disulfide bonds in the carboxy-terminal ends of the A alpha- and gamma-chains and the symmetrical disulfide bonds at gamma 8Cys, gamma 9Cys, and A alpha 28Cys. We studied the loss of thrombin clottability that followed limited reduction and the increase in the susceptibility of the fibrinogen A alpha 19-A alpha 20 bond to hydrolysis by thrombin. Using differential scanning calorimetry, we show that the extent of unfolding and denaturation of specific domains following limited reduction is small. Heat absorption peaks corresponding to the melting of the major regions of compact structure give high calorimetric enthalpies, as in untreated nonreduced fibrinogen, indicating that substantial regions of native structure are still present in partially reduced fibrinogen. Thrombin releases fibrinopeptide A at an identical rate as in nonreduced fibrinogen while fibrinopeptide B release is slower. Sedimentation velocity studies show that thrombin treatment leads to complex formation; however, gelation does not occur. Amino-terminal analysis indicates that the second thrombin cleavage in the A alpha-chain at A alpha 19-A alpha 20 takes place only after fibrinopeptide A release. Thus, the loss of clottability appears to result from perturbation of carboxy-terminal polymerization sites, probably a consequence of gamma 326Cys-gamma 339Cys intrachain disulfide bond cleavage. The thrombin-treated partially reduced fibrinogen remains soluble in buffered saline and fully expresses at least one epitope, B beta 15-21, unique to fibrin. Furthermore, this nonclottable form accelerates the tissue plasminogen activator dependent conversion of plasminogen to plasmin.     

Subunit interaction sites between the regulatory and catalytic subunits of cAMP-dependent protein kinase. Identification of a specific interchain disulfide bond

The catalytic (C) subunit and the type II regulatory (RII) subunit of cAMP-dependent protein kinase can be cross-linked by interchain disulfide bonding. This disulfide bond can be catalyzed by cupric phenanthroline and also can be generated by a disulfide interchange using either RII-subunit or C-subunit that has been modified with either 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or N-4(azidophenylthio)phthalimide (APTP). When the 2 cysteine residues of the C-subunit are reacted with DTNB prior to incubation with the RII-subunit, interchain disulfide bonding occurs. Similar observations are seen with C-subunit that had been modified with APTP. Interchain disulfide bonds also form when the RII-subunit is modified with DTNB prior to incubation with the C-subunit. The presence of cAMP facilitates this cross-linking while autophosphorylation of the RII-subunit retards the rate at which the interchain disulfide bond forms. Interchain disulfide bonds also form spontaneously when the RII-subunit and the C-subunit are dialyzed at pH 8.0 in the absence of reducing agents. The specific amino acid residues that participate in intersubunit disulfide bonding have been identified as Cys-97 in the RII-subunit and Cys-199 in the C-subunit. Based on the sequence homologies of the RII-subunit with other kinase substrates and on the proximity of Cys-97 to the catalytic site, a model is proposed in which the autophosphorylation site of the RII-subunit occupies the substrate-binding site in the holoenzyme. The model also proposes that this same site may be occupied by the region flanking Cys-199 in the C-subunit when the C-subunit is dissociated.     

A comparison of the cyclic nucleotide-dependent protein kinases using chemical cleavage at tryptophan and cysteine

cGMP-dependent protein kinase (G-kinase) and the regulatory subunit of type I (RI) cAMP-dependent protein kinase (A-kinase) both contain a phosphorylation site located near the NH2 terminus of each enzyme. These sites can be utilized as convenient markers for the determination of the position of an amino acid residue susceptible to either chemical or enzymatic digestion. Using the tryptophan-specific reagent, N-chlorosuccinimide, the approximate location along the polypeptide chain of six reactive tryptophans in G-kinase and three reactive residues in RI were identified. Similarly, cleavage with cyanide was used to locate free and disulfide-bonded cysteines in both proteins. The approximate positions of nine cysteines in G-kinase were determined along with the location of the interchain disulfide bond and an intrachain disulfide bond. RI was found to contain three cyanide-reactive cysteines, two of which are involved in interchain disulfide bonding. A comparison of the positions of the cysteines and tryptophans determined by chemical cleavage in G-kinase and RI, with the positions of cysteine and tryptophan in the known sequence of the type II A-kinase, support the structural relationships between these enzymes. Comparison with subsequently reported primary sequences of all three enzymes indicates the limits of precision of this chemical cleavage procedure.     

Location of intrachain disulfide bonds in the VP5* and VP8* trypsin cleavage fragments of the rhesus rotavirus spike protein VP4.

Because the rotavirus spike protein VP4 contains conserved Cys residues at positions 216, 318, 380, and 774 and, for many animal rotaviruses, also at position 203, we sought to determine whether disulfide bonds were structural elements of VP4. Electrophoretic analysis of untreated and trypsin-treated rhesus rotavirus (RRV) and simain rotavirus SA11 in the presence and absence of the reducing agent dithioerythritol revealed that VP4 and its cleavage fragments VP5* and VP8* possessed intrachain disulfide bonds. Given that the VP8* fragments of RRV and SA11 contain only two Cys residues, those at positions 203 and 216, these data indicated that these two residues were covalently linked. Electrophoretic examination of truncated species of VP4 and VP4 containing Cys-->Ser mutations synthesized in reticulocyte lysates provided additional evidence that Cys-203 and Cys-216 in VP8* of RRV were linked by a disulfide bridge. VP5* expressed in vitro was able to form a disulfide bond analogous to that in the VP5* fragment of trypsin-treated RRV. Analysis of a Cys-774-->Ser mutant of VP5* showed that, while it was able to form a disulfide bond, a Cys-318-->Ser mutant of VP5* was not. These results indicated that the VP4 component of all rotaviruses, except B223, contains a disulfide bond that links Cys-318 and Cys-380 in the VP5* region of the protein. This bond is located between the trypsin cleavage site and the putative fusion domain of VP4. Because human rotaviruses lack Cys-203 and, hence, unlike many animal rotaviruses cannot possess a disulfide bond in VP8*, it is apparent that VP4 is structurally variable in nature, with human rotaviruses generally containing one disulfide linkage and animal rotaviruses generally containing two such linkages. Considered with the results of anti-VP4 antibody mapping studies, the data suggest that the disulfide bond in VP5* exists within the 2G4 epitope and may be located at the distal end of the VP4 spike on rotavirus particles.     

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.     

Expression and analysis of COOH-terminal deletions of the human thrombospondin molecule

Thrombospondin (TSP) is a homotrimeric extracellular glycoprotein with a subunit molecular mass of 140 kD. The subunits have a modular or domain-like structure and are held together by interchain disulphide bonds. A number of domains have been identified including those for the binding of collagen, fibrinogen, and heparin. Due to the trimeric form of the TSP molecule, the various domains are trivalent in nature and this contributes to the ability of TSP to mediate cell-substrate interactions. Indeed, TSP has recently been shown not only to promote cell adhesion but also to be intimately involved in cell growth and migration. The adhesive function of TSP is attributable to the "solid-phase" or matrix-bound form of the molecule. There is some evidence that the heparin-binding domain mediates incorporation of soluble TSP into the insoluble matrix form. The heparin-binding domain of TSP is a compact globular amino-terminal moiety that contains two clusters of basic amino acids and a single intrachain disulphide bond. To delineate the role of the heparin-binding domain in matrix assembly and to define further the precise region of interchain disulphide bonding that results in trimer formation, we have expressed deleted forms of the cDNA encoding TSP in SV-40-transformed. African green monkey kidney cells. The proteins synthesized from the various deleted TSP cDNAs were examined for (a) secretion into the culture medium and incorporation into the extracellular matrix; (b) binding to heparin-Sepharose; (c) immunoprecipitability by a conformation-specific monoclonal antibody; and (d) ability to form trimers. This analysis allowed us to draw the following conclusions. (a) A 218 amino acid NH2-terminal protein that preserves the intrachain disulphide bridge of the heparin-binding domain is capable of binding to heparin-Sepharose and incorporating into the extracellular matrix. (b) A shorter 164 amino acid NH2-terminal peptide that does not contain the intrachain disulphide bridge of the heparin-binding domain is neither able to bind to heparin-Sepharose nor able to incorporate into the extracellular matrix. (c) The region of interchain disulphide bridging necessary for trimer assembly resides within a cluster of seven cysteine residues immediately adjacent to the heparin-binding domain.