Complex of pregnancy-associated plasma protein-A and the proform of eosinophil major basic protein. Disulfide structure and carbohydrate attachment

Pregnancy-associated plasma protein-A (PAPP-A) is a metzincin superfamily metalloproteinase responsible for cleavage of insulin-like growth factor-binding protein-4, thus causing release of bound insulin-like growth factor. PAPP-A is secreted as a dimer of 400 kDa but circulates in pregnancy as a disulfide-bound 500-kDa 2:2 complex with the proform of eosinophil major basic protein (pro-MBP), recently shown to function as a proteinase inhibitor of PAPP-A. Except for PAPP-A2, PAPP-A does not share global similarity with other proteins. Three lin-notch (LNR or LIN-12) modules and five complement control protein modules (also known as SCR modules) have been identified in PAPP-A by sequence similarity with other proteins, but no data are available that allow unambiguous prediction of disulfide bonds of these modules. To establish the connectivities of cysteine residues of the PAPP-A.pro-MBP complex, biochemical analyses of peptides derived from purified protein were performed. The PAPP-A subunit contains a total of 82 cysteine residues, of which 81 have been accounted for. The pro-MBP subunit contains 12 cysteine residues, of which 10 have been accounted for. Within the 2:2 complex, PAPP-A is dimerized by a single disulfide bond; pro-MBP is dimerized by two disulfides, and each PAPP-A subunit is connected to a pro-MBP subunit by two disulfide bonds. All other disulfides are intrachain bridges. We also show that of 13 potential sites for N-linked carbohydrate substitution of the PAPP-A subunit, 11 are occupied. The large number of disulfide bonds of the PAPP-A.pro-MBP complex imposes many restraints on polypeptide folding, and knowledge of the disulfide pattern of PAPP-A will facilitate structural studies based on recombinant expression of individual, putative PAPP-A domains. Furthermore, it will allow rational experimental design of functional studies aimed at understanding the formation of the PAPP-A.pro-MBP complex, as well as the inhibitory mechanism of pro-MBP.     

Structural disulfide bonds in the Bacillus thuringiensis subsp. israelensis protein crystal.

We examined disulfide bonds in mosquito larvicidal crystals produced by Bacillus thuringiensis subsp. israelensis. Intact crystals contained 2.01 X 10(-8) mol of free sulfhydryls and 3.24 X 10(-8) mol of disulfides per mg of protein. Reduced samples of alkali-solubilized crystals resolved into several proteins, the most prominent having apparent molecular sizes of 28, 70, 135, and 140 kilodaltons (kDa). Nonreduced samples contained two new proteins of 52 and 26 kDa. When reduced, both the 52- and 26-kDa proteins were converted to 28-kDa proteins. Furthermore, both bands reacted with antiserum prepared against reduced 28-kDa protein. Approximately 50% of the crystal proteins could be solubilized without disulfide cleavage. These proteins were 70 kDa or smaller. Solubilization of the 135- and 140-kDa proteins required disulfide cleavage. Incubation of crystals at pH 12.0 for 2 h cleaved 40% of the disulfide bonds and solubilized 83% of the crystal protein. Alkali-stable disulfides were present in both the soluble and insoluble portions. The insoluble pellet contained 12 to 14 disulfides per 100 kDa of protein and was devoid of sulfhydryl groups. Alkali-solubilized proteins contained both intrachain and interchain disulfide bonds. Despite their structural significance, it is unlikely that disulfide bonds are involved in the formation or release of the larvicidal toxin.     

Domain structure of fibronectin and its relation to function. Disulfides and sulfhydryl groups.

Hamster cell fibronectin is a glycoprotein consisting of two 230,000-dalton subunits in a disulfide-bonded dimer. The molecule is composed of domains which can be separated by partial proteolytic cleavage. The carbohydrates, disulfide bonds, and a single free sulfhydryl group per chain are distributed nonuniformly among these regions. All the interchain disulfides are within 10,000 daltons of the end of the molecule and are removed by mild proteolysis which also generates 200,000- and 25,000-dalton fragments which do not contain interchain disulfides. The 200,000-dalton fragment contains all or most of the carbohydrate side chains, and the free sulfhydryl group, but is relatively poor in cystine. The 25,000-dalton fragment is carbohydrate-free and cystine-rich but has no free sulfhydryl groups. There is heterogeneity in carbohydrate content among the monomeric chains of intact fibronectin and the 200,000-dalton fragments. The gelatin binding site of fibronectin is in the 200,000 fragment. Intact disulfide bonds are required for binding of fibronectin to cells and to gelatin and blockage of the free sulfhydryl groups prevents binding of fibronectin to cells, suggesting that intermolecular disulfide bonding may be important.     

Role of the pro-α2(I) COOH-terminal region in assembly of type I collagen: Disruption of two intramolecular disulfide bonds in pro-α2(I) blocks assembly of type I collagen

Collagen biosynthesis is a complex process that begins with the association of three procollagen chains. A series of conserved intra- and interchain disulfide bonds in the carboxyl-terminal region of the procollagen chains, or C-propeptide, has been hypothesized to play an important role in the nucleation and alignment of the chains. We tested this hypothesis by analyzing the ability of normal and cysteine-mutated pro-α2(I) chains to assemble into type I collagen heterotrimers when expressed in a cell line (D2) that produces only endogenous pro-α1(I). Pro-α2(I) chains containing single or double cysteine mutations that disrupted individual intra- or interchain disulfide bonds were able to form pepsin resistant type I collagen with pro-α1(I), indicating that individual disulfide bonds were not critical for assembly of the pro-α2(I) chain with pro-α1(I). Pro-α2(I) chains containing a triple cysteine mutation that disrupted both intrachain disulfide bonds were not able to form pepsin resistant type I collagen with pro-α1(I). Therefore, disruption of both pro-α2(I) intrachain disulfide bonds prevented the production and secretion of type I collagen heterotrimers. Although none of the individual disulfide bonds is essential for assembly of the procollagen chains, the presence of at least one intrachain disulfide bond may be necessary as a structural requirement for chain association or to stabilize the protein to prevent intracellular degradation. J.Cell. Biochem. 71:233–242, 1998. © 1998 Wiley-Liss, Inc.     

Disulfide bonds are localized within the short consensus repeat units of complement regulatory proteins: C4b-binding protein

Several plasma and membrane proteins belong to a superfamily of structurally related proteins that contain internal homology of a variable number (2-30) of repeating units. Each SCR (short consensus repeat) unit is approximately 60 amino acid residues in length, with the positions of 1 Trp, 2 Pro, and 4 Cys residues being conserved. The aim of this study was to provide experimental evidence that each SCR may exist as an independent structural domain maintained by disulfide bonds. The well-characterized C4b-binding protein (C4BP) with eight SCR units in each of its seven identical chains was chosen for this study. Analysis of the disulfide-bonding pattern indicated that intrachain disulfide bonds may be localized within each SCR unit, with the first and third and the second and fourth half-cystines in each unit being linked. This pattern of disulfides may confer to C4BP (and to other structurally related proteins) a conformation which apparently allows the assembly of the SCR units (4-30) in a tandem fashion. Such an arrangement of the polypeptide chain(s) may explain, in part, the elongated shape of these protein molecules. The structural motif of the SCR units of C4BP is discussed in relation to those previously described for the type II domain of fibronectin and the kringle structure present in various proteins of the coagulation system.     

Suppression of sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample preparation artifacts for analysis of IgG4 half-antibody

Human IgG4 subtype antibodies have often been reported to have a significant portion (5-50%) of a heavy chain-light chain dimer ("half-antibody") on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in which the heavy chain is not covalently linked through the hinge disulfides to another heavy chain. We demonstrate here that there can be artifactual sources of half-antibody. One occurred during SDS-PAGE sample preparation where rapid disulfide scrambling was initiated by preexisting free sulfhydryls in the monoclonal antibody (mAb) and by free sulfhydryl produced by destruction of disulfide bonds during heating. Inclusion of N-ethylmaleimide in the sample buffer prevented the disulfide scrambling. Presumably, cyclization of the flexible IgG4 hinge during this disulfide scrambling leads to the preferential separation of heavy chains. A second condition producing half-antibody was reoxidation after exposure to reductant, where 46% of the antibody was trapped in the intrachain disulfide form. The amount of half-antibody was reduced to 4% by reoxidation in the presence of a mixture of oxidized and reduced glutathione. When the improved sample preparation conditions were used, IgG4 mAb freshly isolated from cells contained 4.5-15% half-antibody, indicating that equilibration of the interchain and intrachain hinge disulfide pairing was not always attained in cells.     

Co-translational modification of nascent immunoglobulin heavy and light chains

We have investigated the in vivo co-translational covalent modification of nascent immunoglobulin heavy and light chains. Nascent polypeptides were separated from completed polypeptides by ion-exchange chromatography of solubilized ribosomes on QAE-Sephadex. First, we have demonstrated that MPC 11 nascent heavy chains are quantitatively glycosylated very soon after the asparaginyl acceptor site passes through the membrane into the cisterna of the rough endoplasmic reticulum. Nonglycosylated completed heavy chains of various classes cannot be glycosylated after release from the ribosome, due either to rapid intramolecular folding and/or intermolecular assembly, which cause the acceptor site to become unavailable for the glycosylation enzyme. Second, we have shown that the formation of the correct intrachain disulfide loop within the first light chain domain occurs rapidly and quantitatively as soon as the appropriate cysteine residues of the nascent light chain pass through the membrane into the cisterna of the endoplasmic reticulum. The intrachain disulfide loop in the second or constant region domain of the light chain is not formed on nascent chains, because one of the cysteine residues involved in this disulfide bond does not pass through the endoplasmic reticulum membrane prior to chain completion and release from the ribosome. Third, we have demonstrated that some of the initial covalent assembly (formation of interchain disulfide bonds) occurs on nascent heavy chains prior to their release from the ribosome. The results are consistent with the pathway of covalent assembly of the cell line, in that completed light chains are assembled onto nascent heavy chains in MPC 11 cells (IgG₂b), where a heavy-light half molecule is the major initial covalent intermediate; and completed heavy chains are assembled onto nascent heavy chains in MOPC 21 cells (IgG₁), where a heavy chain dimer is the major initial disulfide linked intermediate.     

Intrachain disulfide bond in the core hinge region of human IgG4.

IgG is a tetrameric protein composed of two copies each of the light and heavy chains. The four-chain structure is maintained by strong noncovalent interactions between the amino-terminal half of pairs of heavy-light chains and between the carboxyl-terminal regions of the two heavy chains. In addition, interchain disulfide bonds link each heavy-light chain and also link the paired heavy chains. An engineered human IgG4 specific for human tumor necrosis factor-alpha (CDP571) is similar to human myeloma IgG4 in that it is secreted as both disulfide bonded tetramers (approximately 75% of the total amount of IgG) and as tetramers composed of nondisulfide bonded half-IgG4 (heavy chain disulfide bonded to light chain) molecules. However, when CDP571 was genetically engineered with a proline at residue 229 of the core hinge region rather than serine, CDP571 (S229P), or with an IgG1 rather than IgG4 hinge region, CDP571(gamma 1), only trace amounts of nondisulfide bonded half-IgG tetramers were observed. Trypsin digest reversephase HPLC peptide mapping studies of CDP571 and CDP571(gamma 1) with on-line electrospray ionization mass spectroscopy supplemented with Edman sequencing identified the chemical factor preventing inter-heavy chain disulfide bond formation between half-IgG molecules: the two cysteines in the IgG4 and IgG1 core hinge region (CPSCP and CPPCP, respectively) are capable of forming an intrachain disulfide bond. Conformational modeling studies on cyclic disulfide bonded CPSCP and CPPCP peptides yielded energy ranges for the low-energy conformations of 31-33 kcal/mol and 40-42 kcal/mol, respectively. In addition, higher torsion and angle bending energies were observed for the CPPCP peptide due to backbone constraints caused by the extra proline. These modeling results suggest a reason why a larger fraction of intrachain bonds are observed in IgG4 rather than IgG1 molecules: the serine in the core hinge region of IgG4 allows more hinge region flexibility than the proline of IgG1 and thus may permit formation of a stable intrachain disulfide bond more readily.     

Amino acid sequence of the light chain derived from a rabbit anti-p-azobenzoate antibody of restricted heterogeneity

The amino acid sequence of the light chain from a specifically purified rabbit (No. 2717) anti-p-azobenzoate antibody preparation (b4 allotype) of restricted heterogeneity has been determined. This light chain is composed of 216 residues, including seven half-cystine residues located at positions 23, 80, 88, 134, 171, 194 and 216. Three intrachain disulfide bonds appear to be present in contrast to only two disulfide bonds as has been so far described for Bence Jones protein and light chains of human and mouse. This light chain was sequenced by isolating the tryptic peptides, sequencing the peptides and establishing their order within the molecule. Unambiguous identification of the overlaps was achieved by taking into account the partially characterized tryptic peptides from citraconic anhydride-treated light chains and chymotryptic and peptic peptides from digests of both untreated and citraconylated light chains. Comparison of this amino acid sequence with the amino acid sequence of the car?ylterminal half of the b4 light chains from unimmunized rabbits reveals differences at positions 165, 166, 169 and 176 indicating the existence of more than one sequence in the b4 “constant” region. There is substantial sequence homology between the variable half of 2717 light chain and human Bence Jones protein. Indeed, 46 positions in the V region (42%) are occupied by the same residues in this light chain and in human subgroup V_κIII.     

Assignment of interchain disulfide bonds in platelet-derived growth factor (PDGF) and evidence for agonist activity of monomeric PDGF.

Platelet-derived growth factor (PDGF) is a dimeric factor stabilized by disulfide bonds. Using an approach involving partial reduction of PDGF, we have identified the 2nd and 4th cysteine residues in the PDGF chains as the cysteine residues forming interchain disulfide bonds. Analysis of PDGF mutants in which the 2nd and 4th cysteine residues were mutated to serine residues revealed that the disulfide bonds are arranged in a cross-wise manner, with the 2nd cysteine residue in one chain being linked to the 4th cysteine residue in the other. A PDGF B-chain mutant, in which both the 2nd and 4th cysteine residues were substituted with serine residues, migrated as a monomer in sodium dodecyl sulfate gel electrophoresis and retained receptor binding activity. When analyzed in receptor dimerization and autophosphorylation assays, this mutant showed agonistic activity. Thus, structural information has been obtained that will allow the large scale production of properly folded monomeric PDGF, as well as design of specific PDGF heterodimers.     

The interdomain disulfide bond of a homogeneous rabbit pneumococcal antibody light chain.

Rabbit light chain 3315, prepared from a homogeneous antipneumococcal antibody, was subjected to hydrolysis by pepsin without prior reduction and alkylation of the intrachain disulfide bonds. Gel filtration of the hydrolysate on Sephadex G-10, G-15, and G-25 and ion exchange chromatography on SP-Sephadex yielded several disulfide bridge peptides. These were fully reduced and alkulated and sequenced by Edman degradation. The peptides were located in the light chain sequence determined in independent studies from our laboratory. The half-cystine residues in this KB rabbit chain are located at positions 23, 80, 88, 134, 171, 194, and 214. The extra disulfide bridge extends between residues 80 and 171, thus joining the variable and constant domains. This is consistent with x-ray diffraction crystallographic studies showing that the corresponding residues in human light chains are separated by a distance compatible with disulfide bond formation.     

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.     

Different susceptibility of inter- and intra-chain disulfide bonds to reductive cleavage in native fibronectin and effect of their cleavage on conformation

The two thread-like subunits (Mr approximately equal to 250 000) of the multidomain protein fibronectin are connected by a pair of inter-chain disulfide bridges in their C-terminal regions. In addition each chain contains 29 intra-chain disulfide bonds which are located in 12 type I and 2 type II structural domains in the N-terminal and C-terminal regions of the strands. The 15 to 17 type III domains in the central portion of the strands do not contain disulfide bonds. The susceptibility of inter-chain disulfide bonds to 10mM 1,4-dithiothreitol at pH 7.8 as quantitated by the rate of reductive cleavage of fibronectin into its subunits was found to be only 8-fold larger than that of the intra-chain bonds. Consequently at 90% completion of chain separation 30% of the intra-chain disulfides are also cleaved. The rate of inter-chain disulfide cleavage was found to be identical for fibronectin and a 140-kDa fragment comprising the C-terminal portions of the two subunits. This shows that the relatively high protection of the inter-chain disulfide bonds must originate from interactions between C-terminal domains which are probably also responsible for the V shaped arrangement of the two subunit strands. Changes of circular dichroism and thermal transition profiles for fibronectin and its C-terminal 140-kDa fragment indicated that already partial reduction of the intra-chain disulfide bonds alters the conformations of type I and II domains without affecting the type III domains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complete amino acid sequence of a papain-solubilized human histocompatibility antigen, HLA-B7. 2. Sequence determination and search for homologies

The complete amino acid sequence of the papain-solubilized heavy chain of a human histocompatibility antigen (HLA-B7) has been elucidated. It consists of a polypeptide of 271 residues (31 333 daltons). A single glycan moiety is attached to an asparagine residue at position 86 by an N-glycosidic bond. Two intrachain disulfide bonds, arranged linearly, involve half-cystine residues at positions 101 and 164 and at positions 203 and 259. They form two loops of 62 and 55 residues, respectively, separated by 38 residues. Computer analysis of the sequence suggests the existence of internal homology between the amino-terminal portion (residues 1--90) and the region of the first disulfide loop (residues 91--180). There is a significant homology between the second disulfide loop region of the chain (residues 182-271) and immunoglobulin (Ig) constant domains and beta 2-microglobulin [Orr, H. T., Lancet, D., Robb, R. J., López de Castro, J. A., & Strominger, J. L. (1979A) Nature (London) (in press)]. However, no such homology to Ig is apparent in the amino-terminal or in the first disulfide loop regions.     

Membrane immunoglobulins are stabilized by interchain disulfide bonds occurring within the extracellular membrane-proximal domain

Membrane-bound immunoglobulins have, in addition to the transmembrane and cytoplasmic portions, an extracellular membrane-proximal domain (EMPD), absent in the secretory forms. EMPDs of immunoglobulin isotypes alpha, gamma, and epsilon contain cysteines whose role has so far not been elucidated. Using a genetic strategy, we investigated the ability of these cysteines to form disulfide bridges. Shortened versions of human membrane immunoglobulins, depleted of cysteines known to form intermolecular disulfide bonds, were constructed and expressed on the surface of a B-cell line. The resulting membrane proteins contain a single chain fragment of variable regions (scFv) linked to the dimerizing domain from the immunoglobulin heavy chains (CH3 for alpha and gamma or CH4 for epsilon isotypes), followed by the corresponding EMPD and the transmembrane and cytoplasmic domains. The two functional membrane versions of the epsilon chain, containing the short and long EMPD, were analyzed. Our results show that the single cysteine within alpha1L and gamma1 EMPD and the short version of epsilon EMPD form an interchain disulfide bond. Conversely, the cysteine resident in the epsilon transmembrane domain remains unreacted. epsilon-long EMPD contains four cysteines; two are involved in interchain bonds while the remaining two are likely forming an intrachain bridge. Expression of a full-length membrane epsilon heavy chain mutant, in which Cys(121) and Cys(209) within domain CH2 (involved in interchain bridges) were mutated to alanines, confirmed that, within the complete IgE, EMPD cysteines form interchain disulfide bonds. In conclusion, we unveil evidence for additional covalent stabilization of membrane-bound immunoglobulins.     

Recombinant 70-kDa protein from the amino-terminal region of rat fibronectin inhibits binding of fibronectin to cells and bacteria

Binding of fibronectin to substrate-attached cells and to Staphylococcus aureus is mediated by the amino-terminal 70-kDa portion of fibronectin. The 70-kDa amino-terminus is composed of nine type I and two type II internal homology units, each containing two intrachain disulfide bonds. The exact structural features of the 70-kDa amino-terminus that are necessary for binding to cells and bacteria are not known. We characterized a recombinant 70-kDa protein from the amino-terminus of rat fibronectin using a baculovirus expression system. Recombinant 70-kDa (r70kDa) protein was easily purified in high amounts from the conditioned medium by affinity chromatography on gelatin-agarose. Secretion was much less when N-linked glycosylation was blocked by tunicamycin. Like the native fragment, the r70kDa protein contains intrachain disulfide bonds. In addition, the r70kDa protein was indistinguishable from the nonrecombinant 70-kDa fragment in its ability to compete for binding sites on fibroblasts and S. aureus. Thus, the r70kDa protein retains the important functional characteristics of the native fragment. This expression system is well adapted to studying the structural features important for the interaction of 70-kDa protein with cells.     

Detection of disulfide bonds in bovine brain tubulin and their role in protein folding and microtubule assembly in vitro: a novel disulfide detection approach

Cysteine residues in tubulin are actively involved in regulating ligand interactions and microtubule formation both in vivo and in vitro. These cysteine residues are sensitive reporters in determining the conformation of tubulin. Although some of the cysteines are critical in modulating drug binding and microtubule assembly, it is not clear how many of these normally exist as disulfides. The controversy regarding the disulfide bonds led us to develop a disulfide detection assay to reexamine the presence of the disulfide linkages in purified alphabeta tubulin and explore their possible biological functions in vitro. The accessible cysteine residues in alphabeta tubulin were alkylated with an excess of iodoacetamide to prevent artifactual generation of disulfide linkages in tubulin. After removal of excess iodoacetamide, tubulin was unfolded in 8 M urea. Half of the unfolded tubulin was treated with dithiothreitol to reduce any disulfide bonds present. The aliquots were then treated with iodo[(14)C]acetamide and the incorporation of radioactivity was measured. We also used the same approach to detect the disulfide linkages in the tubulin in a whole-cell extract. We found in both cases that the samples which were not treated with dithiothreitol had little or no incorporation of iodo[(14)C]acetamide, while the others that were treated with dithiothreitol had significant amounts of (14)C incorporation into tubulin. Moreover, the reduction of the disulfide linkages in tubulin resulted in inhibition of microtubule assembly (29-54%) and markedly affected refolding of the tubulin from both an intermediate and a completely unfolded state. All these data therefore suggest that tubulin has intrachain disulfide bonds in the alpha- and beta-subunits and that these disulfides assist in correct refolding of tubulin from the intermediate unfolded state or help to recover the hydrophobic domains from the completely unfolded state. These disulfides also regulate microtubule assembly and the stability of tubulin in vitro. Our results suggest that tubulin disulfides may play a role in tubulin folding and that thiol-disulfide exchange in tubulin could be a key regulator in microtubule assembly and dynamics of tubulin in vivo.     

Novel S-S loops in the giant hemoglobin of Tylorrhynchus heterochaetus

The extracellular hemoglobin of the polychaete Tylorrhynchus heterochaetus is a "giant" multisubunit protein consisting of two types of subunits: a "monomeric" chain (chain I) and a disulfide bonded "trimer" of chains IIA, IIB, and IIC. We have reported the complete amino acid sequences of all four chains (Suzuki, T., and Gotoh, T. (1986) J. Biol. Chem. 261, 9257-9267). The sites of disulfide bonds in the trimer have now been determined. In the trimer, there are two interchain disulfide bonds between chains IIA and IIC, and IIB and IIC, respectively. In addition, each of the four chains, I, IIA, IIB, IIC, has an intrachain disulfide bond. Thus, according to our "192-chain" model (Suzuki, T., and Gotoh, T. (1986) J. Mol. Biol. 190, 119-123), there are 288 disulfide bonds in Tylorrhynchus hemoglobin. Digital image processing of scanning transmission electron micrographs of negatively stained Tylorrhynchus hemoglobin indicated dimensions of 28 x 18 nm.     

Correlation between the exposure of aromatic chromophores at the surface of the Fc domains of immunoglobulin G and their ability to bind complement.

The recognition that certain biological effector functions associated with the Fc region of human IgG are mediated exclusively by either the Cgamma2 or Cgamma3 domains prompted a study of some of the physical properties of the isolated domains in an attempt to correlate these with functional differentiation. The degree of aromatic chromophore exposure of intact Fc and fragments corresponding to the Cgamma2 and Cgamma3 domains were determined by solvent perturbation difference spectroscopy using 20% ethylene glycol. For the monomeric Cgamma2 fragment one of the two tryptophans and all four of the tyrosines were exposed to solvent. In the pFc' fragment, which represented a dimer of two intact Cgamma3 domains, an average of 0.4 of the two tryptophans of 3.3 of the five tyrosines per chain were exposed. These data were consistent with the suggested involvement of tryptophan in complement fixation since Cgamma2 binds C1q but pFc' does not. Several fragments derived from the Cgamma3 region had previously been shown to have differing environments for their aromatic side chains from circular dichroism studies. These fragments have now been shown to exhibit different degrees of chromophore exposure to solvent. Removal of the carboxy-termimal heptapeptide from the intact, Cgamma3 domain resulted in a fragment not only showing a greater exposure of aromatic residues but also having the ability to bind Clq. Our data suggest that the structural requirements for C1Q binding may be quite commonplace within Fc, but tertiary folding limits their expression except in Cgamma2 in the native molecule. The solvent perturbation observed with Fc was somewhat lower than would have been expected from the results with the isolated domains, suggesting that interdomain interactions may result in burial of aromatic residues.     

An extra disulfide bridge in the constant domain of Rana catesbeiana immunoglobulin light chains

The immunoglobulins of the bullfrog Rana catesbeiana are unusual in that, in all classes, the light chains are not disulfide bonded to heavy chains or to other light chains. Moreover, the light chains contain six, rather than the usual five, residues of half-cystine. As none of these half-cystines is in the sulfhydryl form or is alkylated after mild reduction, we suggested that the light chains probably contain three intrachain disulfide bridges. We have now carried out experiments to confirm the existence of an extra intrachain disulfide bridge in Rana catesbeiana light chains and to determine its location. Disulfide bridge assignments were based on 1) isolation and sequence analysis of S-(carboxymethyl)cysteine-containing peptides and 2) isolation, from unreduced light chains, of peptides containing a disulfide bridge. Half-cystine residues were found at positions 134 and 194, and these were shown to be joined in the conserved intradomain disulfide bridge. In addition, we found that a residue of half-cystine, located at the third position from the carboxy-terminus, forms a disulfide bridge with a half-cystine at position 119, near the amino-terminus of the domain, the latter residue replacing a proline that has been found at this position in all other light chains. An intrachain disulfide bridge has not been found at this location in any other light chain.