Role of disulfide bond formation in the folding of human chorionic gonadotropin beta subunit into an alpha beta dimer assembly-competent form.

The malignant trophoblastic cell line JAR was used as a model system to study protein folding in intact cells. We have used this model previously to identify conformational intermediates in the production of an assembly-competent form of the human chorionic gonadotropin beta subunit (Ruddon, R. W., Krzesicki, R. F., Norton, S. E., Beebe, J. S., Peters, B. P., and Perini, F. (1987) J. Biol. Chem. 262, 12533-12540). The earliest biosynthetic precursor of the human chorionic gonadotropin beta subunit detectable in JAR cells pulse labeled for 2 min is p beta 1, a form that lacks half of the six intrachain disulfide bonds observed in the fully processed dimer form of beta and that does not combine with the alpha subunit. p beta 1 is rapidly (t1/2 approximately 4 min) converted into p beta 2, which has a full complement of intrachain disulfide bonds and does combine with the alpha subunit. In this study, we have identified the three late forming disulfide bonds involved in the transition of p beta 1 into the assembly-compete form, p beta 2. The last three disulfide bonds to form are those between cysteines 9 and 90, 23 and 72, and 93 and 100. These were identified in JAR cell lysates that had been pulse labeled with [35S]cysteine for 2 or 5 min followed by trapping of the cysteine thiols with iodoacetic acid before immunopurification of the beta subunit forms. Immunopurified p beta 1 was treated with trypsin under nonreducing conditions to liberate [35S]cysteine-containing peptides from the disulfide-linked beta core polypeptide. These tryptic peptides were then separated by high performance liquid chromatography and sequenced to determine the location of the carboxymethyl-[35S]cysteine residues. The three late forming disulfide bonds are most likely the ones involved in stabilizing the conformation of the beta subunit that is required for combination with alpha to form the biologically functional alpha beta heterodimer.     

Primary structure of human fibrinogen and fibrin. Isolation and partial characterization of chains of fragment D.

Fragment D has been isolated as an apparently single molecular weight species (molecular weight about 100,000) from plasmin digests of humman fibrinogen, using a combination of affinity chromatography on insolubilized "fibrin monomer" and gel filtration. This fragment consists of three chains with molecular weights of 15,000 (Dbeta), 42,500 (Dgamma1) or 39,500 (Dgamma2), and 14,000 (Dalpha) held together by disulfide bonds. The S-carboxymethyl derivatives of the chains have been separated by gel filtration and ion exchange chromatography, and their identity has been confirmed by peptide mapping and immunological analysis. The chain with a molecular weight of 45,000 is a fragment of the Bbeta chain of fibrinogen. The chain derived from the gamma chain of fibrinogen occurred in two molecular forms having molecular weight 42,500 and 39,500. The chain derivative with molecular weight 14,000 is most likely derived from the Aalpha chain of fibrinogen. The chains were characterized by NH2-terminal sequence analysis, amino acid composition, and carbohydrate staining. The two molecular analysis, amino acid composition, and carbohydrate staining. The two molecular forms of the gamma chain appeared to be identical except for an NH2-terminal peptide extension of 23 amino acid residues in the longer chain. The latter has sequences in common with the COOH-terminal part of the gamma chain of the NH2-terminal disulfide knot (BROMBACK, B., BRONDAHL, N. J., HESSEL, B., IWANAGA, S., and WALLEN, P. (1973) J. Biol. Chem. 248, 5806-5820); its NH2-terminal residue being Ala-63 of the gamma chain of fibrinogen.     

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.     

Electron transfer in monomeric forms of beef and shark heart cytochrome c oxidase

Beef heart cytochrome c oxidase is dimeric in reconstituted membranes and in nonionic detergents at physiological pH [Henderson, R., Capaldi, R. A., & Leigh, J. (1977) J. Mol. Biol. 112, 631; Robinson, N.C., & Capaldi, R. A. (1977) Biochemistry 16, 375], raising the possibility that this aggregation state is a prerequisite for enzymatic activity. A procedure for dissociating the enzyme into monomers is presented. This involves treating the protein with high concentrations of Triton X-100 at pH 8.5. The electron transfer activity of the monomer is comparable to that of the dimer under identical assay conditions. The beef heart cytochrome c oxidase monomer was found to be heterogeneous in hydrodynamic studies, probably due to dissociation of associated polypeptides, including subunit III. Monomer molecular weights in the range 129 000-160 000 were obtained. Previous studies have indicated that shark heart cytochrome c oxidase is monomeric under physiological conditions. Sedimentation equilibrium studies reported here confirm this. The elasmobranch enzyme, with a similar polypeptide composition to that of beef enzyme, was determined to have a molecular weight of 158 000.     

DNA-mediated gene transfer of a human cell surface 170-kilodalton glycoprotein. Evidence for association with an endogenous murine protein

We have previously reported the identification and characterization of two related human cell surface protein complexes, very common antigens 1 and 2 (VCA-1, VCA-2) (Kantor, R. R. S., Mattes, M. J., Lloyd, K. O., Old, L. J., and Albino, A. P. (1987) J. Biol. Chem. 262, 15158-15165). We now report the transfection of DNA sequences encoding the 170-kilodalton heterodimer of VCA-2 from human SK-RC-41 renal cancer cells to B78H1 mouse melanoma cells. B78H1 cells were cotransfected with high molecular weight renal cancer DNA and a plasmid vector containing the neomycin resistance gene. Antibiotic-resistant transfectants were screened for the expression of the 170-kDa heterodimer with mouse monoclonal antibody (mAb) J143. Analysis of mAb J143-positive (J143+) transfectants showed that they expressed a 170-kDa heterodimer with an identical molecular weight, isoelectric point, two-dimensional peptide map, and spatial orientation of surface-exposed epitopes to the homologous 170-kDa species seen in human donor cells. The 170-kDa heterodimer in SK-RC-41 cells is associated with a 140-kDa (designated 140(1] polypeptide to form the VCA-2 complex. The 170-kDa complex and the 140(1)-kDa polypeptides are encoded by genes located on different human chromosomes. J143+ transfectants display a molecule of 140 kDa associated with the 170-kDa complex which is biochemically similar, but non-identical, to the human 140(1)-kDa polypeptide on VCA-2. This evidence supports our interpretation that the transfected human 170-kDa heterodimer associates with a murine counterpart of the human 140(1)-kDa polypeptide in J143+ transfectants.     

Structural studies of apolipoprotein B: physical properties of the protein in guanidine hydrochloride

Apolipoprotein B was isolated from human plasma low-density-lipoprotein without precipitation by diethyl ether/ethanol extraction of the protein in 6 M guanidine hydrochloride. The physical properties of this protein, which contained a residuum of approximately 7% phospholipid, were examined in 6 M guanidine solution under reducing conditions. The circular dichroism spectrum was indistinguishable from that of a random coil protein. Sedimentation equilibrium analyses of apolipoprotein B by the meniscus depletion method of Yphantis (1984, Biochemistry 3, 297-317) were complicated by heterogeneity and nonideality despite the low concentrations employed. 63 analyses of the weight average (Mw) and z average (Mz) molecular weight were made on the apolipoprotein B from 12 subjects. The Mw observed was a function of initial concentration, rotor speed, and a heterogeneity index (Mz/Mw). Multiple linear regression of apolipoprotein B molecular mass against these parameters suggested that an Mw of 540,000 +/- 110,000 would be observed under apparently ideal and homogeneous conditions. The sedimentation coefficient and intrinsic viscosity of the reduced protein at 25 degrees C in 6 M guanidine were 2.13 S and 116 ml/g, respectively; these values predict molecular weights of 640,000 and 250,000, respectively, if apolipoprotein B was fully denatured into a random coil. Lack of agreement between these estimates and with the sedimentation equilibrium analysis can best be explained by compactness of structure and incomplete denaturation to a random coil state. Furthermore, an irreversible temperature dependence of apolipoprotein B reduced viscosity indicated that residual structure remained in solutions of 6 M guanidine hydrochloride/20 mM dithiothreitol. Taken together, the physical data demonstrate that apolipoprotein is a single polypeptide of approximately 540 kDa, whose structure resists denaturation under conditions where most proteins exist as random coils.     

Lymphoma-selective antibody Lym-1 recognizes a discontinuous epitope on the light chain of HLA-DR10

 The selectivity of Lym-1 for malignant B lymphocytes makes this monoclonal antibody a promising candidate for the delivery of toxic agents to malignant B cells. The original immunogen used for the development of Lym-1 was Raji Burkitt’s lymphoma cell nuclei [Epstein A. L., Marder R. J., Winter J. N., Stathopoulos E., Chen F. M., Parker J. W., Taylor C. R. (1987) Cancer Res 47: 830]. The Lym-1 antigen was characterized at that time as a polymorphic HLA-DR variant. We prepared an affinity column using immobilized Lym-1 to isolate the Lym-1 antigen from Raji cell lysate. Immunological characterization of the immunoaffinity-purified Lym-1 antigen on Western blots led to the conclusion that the antigen is the β chain of HLA-DR10. This was confirmed by Edman sequencing of the isolated polypeptide chain. Western blots further show that the Lym-1 epitope is only recognized if the β chain disulfide bonds are intact. These results imply that Lym-1 binds a discontinuous epitope on the β chain of HLA-DR10. Received: 22 February 1996 / Accepted: 28 June 1996     

Partial Purification and Some Properties of the Staphylococcus aureus Cytoplasmic Nitrate Reductase

The cytoplasmic nitrate reductase in heme mutant H-14 of Staphylococcus aureus was partially purified by steps which included ammonium sulfate fractionation and chromatography on Bio-Gel A 1.5m and ion-exchange columns. The active fractions from the ion-exchange columns showed two forms of the enzyme upon electrophoresis in nondenaturing gels of polyacrylamide; these corresponded to proteins of R(f) 0.16 and 0.28. Each form contained a predominant polypeptide of molecular weight 140,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The R(f) 0.16 form contained another major polypeptide of molecular weight 57,000, but the R(f) 0.28 form contained several other polypeptides. The sedimentation properties of the enzyme were examined after partial purification on Bio-Gel A 1.5m. In sucrose gradients containing Triton X-100 the enzyme sedimented as a homogeneous peak with an estimated molecular weight of 225,000; without detergent a heterogeneous profile was observed of molecular weight greater than 250,000. Treatment of the enzyme with trypsin increased the specific activity, and the enzyme sedimented as a homogeneous peak in sucrose gradients without Triton X-100, with an estimated molecular weight of 202,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that trypsin treatment converted the polypeptide of molecular weight 140,000 to a polypeptide of molecular weight 112,000. We conclude that the cytoplasmic nitrate reductase of S. aureus has a large subunit of molecular weight 140,000, which can be modified by trypsin to a polypeptide of molecular weight 112,000 without loss of catalytic activity.     

Antiparallel alignment of the two protomers of the regulatory subunit dimer of cAMP-dependent protein kinase I

The purified type I regulatory subunit of cAMP-dependent protein kinase is a dimeric protein, and the two protomers of the dimer are linked by two interchain disulfide bonds. The disulfide linkages that join these two polypeptide chains have been identified in order to provide a structural basis for the orientation of the two chains in the asymmetric dimer. Disulfide bonds were found to exist exclusively between Cys-16 and Cys-37, and this assignment, thus, establishes a general antiparallel alignment of the two chains. Two other homologous proteins, the type II regulatory subunit and the cGMP-dependent protein kinase also are dimeric proteins. In all three proteins, a relatively small, nonhomologous, amino-terminal segment of the polypeptide chain is essential for maintaining the dimeric aggregation state.     

Subunit structure of extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus and amino acid sequence of the constituent polypeptide chain (IIC)

Tylorrhynchus cyanomethemoglobin reduced with dithiothreitol was separated by chromatofocusing into four heme-containing polypeptide chains (I, IIA, IIB, and IIC) and a non-heme chain (N). The molecular weights of chains IIA-C and N were confirmed to be the same by polyacrylamide gel electrophoresis in sodium dodecyl sulfate on a 10-20% gradient gel. The molecular weight of chain IIC was determined to be 17,415 (including heme) from the amino acid sequence. Chain N constitutes less than 5% of the total protein and has the same NH2-terminal sequence, suggesting that it is derived from chain IIA during the isolation procedure. Tylorrhynchus hemoglobin consists of two types of subunit with molecular weights of 16,327 (chain I) and approximately 50,000, and the latter splits into chains IIA-C in the presence of a reducing agent. On the basis of the accurate value obtained for the molecular mass of chain IIC, it was concluded that the subunit of approximately 50,000 daltons is a trimer of heme-containing chains IIA, IIB, and IIC linked by disulfide bonds. The cysteine residue at position 5 and the arginine at position 10 are conserved in the four heme-containing chains of Tylorrhynchus hemoglobin. The complete sequence of 149 residues of Tylorrhynchus chain IIC was determined. This sequence shows high homology with Tylorrhynchus chain I (Suzuki, T., Takagi, T., and Gotoh, T. (1982) Biochem. Biophys. Acta 708, 253-258) and Lumbricus chain AIII (Garlick, R. L., and Riggs, A. F. (1982) J. Biol. Chem. 257, 9005-9015).     

Human plasma lipoprotein [a]. Structural properties

When lipoprotein [a] was isolated in the presence of the proteolytic inhibitor Trasylol, its apoprotein exhibited one dominant band corresponding to a molecular weight of about 1.2 million when analyzed by electrophoresis on 3.25% sodium dodecyl sulfate-polyacrylamide gels. After chemical reduction, this band was missing but was replaced by two bands, one corresponding to a molecular weight of about 490,000 and the other to a molecular weight of about 645,000. Before treatment with reducing agents, the apolipoprotein [a] and apolipoprotein B immunoreactivities were detectable in the same electrophoretic band, but after reduction the apolipoprotein [a] was demonstrated to be separate from the apolipoprotein B. These results suggest that the apoprotein of lipoprotein [a] is composed of two subunits which are similar in molecular weight and are held together by one or more disulfide bonds. One subunit possesses apolipoprotein [a] and the other apolipoprotein B immunoreactivity. The secondary structure of the apoprotein components within lipoprotein [a] has been studied by circular dichroism and found to differ significantly from the secondary structure of the apoproteins in low density lipoproteins and high density lipoproteins. About 30% alpha-helical structure was measured in lipoprotein [a] compared to 48% in low density lipoproteins and 70% in high density lipoproteins. Lipoprotein [a] exhibited a much higher percentage of disordered structure than either of the other two lipoproteins.     

Protein composition of Lp(a) lipoprotein from human plasma

The apolipoprotein composition of purified human Lp(a) lipoprotein was investigated by SDS--polyacrylamide gel electrophoresis and immunochemically. The lipoprotein contains two different polypeptides. One is identical by its app. Mr of approximately 250 000 and immunologically with apolipoprotein B of LDL (B-100). The other polypeptide has a higher app. Mr (approximately 350 000) and stains strongly with the periodate-Schiff's reagent. This high-Mr glycoprotein contains the specific Lp(a) immunoreactivity but does not react with antibodies against apo B. Apo B and Lp(a)-protein seem to be linked by disulfide bonds in the native lipoprotein. The unreduced detergent delipidized protein moiety from Lp(a) lipoprotein shows a single band of Mr approximately 700 000 in SDS--polyacrylamide gel electrophoresis and the immunoprecipitates formed against anti-Lp(a) and anti-apo B by the unreduced protein show a reaction of immunological identity.     

Amino acid sequence and post-translational modification of stem cell factor isolated from buffalo rat liver cell-conditioned medium.

Stem cell factor (SCF) isolated from culture medium conditioned by Buffalo rat liver cells was subjected to detailed structural analysis. Attempts at direct N-terminal sequencing of the factor indicated that its N terminus is blocked as pyroglutamic acid (Zsebo, K. M., Wypych, J., McNiece, I. K., Lu, H. S., Smith, K. A., Karkare, S. B., Sachdev, R. K., Yuschenkoff, V. N., Birkett, N. C., Williams, L. R., Satyagal, V. N., Bosselman, R. A., Mendiaz, E. A., and Langley, K. E. (1990) Cell 63, 195-201). The removal of the blocking pyroglutamate by pyroglutamate aminopeptidase allowed sequencing of the polypeptide chain to position 47. Stem cell factor was also digested with CNBr, trypsin, Staphylococcus aureus protease (strain V8), and AspN peptidase to generate different sets of peptides that were then separated by reverse-phase high-performance liquid chromatography and sequenced. Sequence of an internal peptide fragment obtained by cleavage of stem cell factor at a single tryptophanyl peptide bond was also obtained. From these analyses, the complete amino acid sequence could be constructed. The factor as isolated is a single polypeptide of 164 or 165 amino acids. The sequence is confirmatory to a sequence deduced from a cDNA sequence and provides important evidence for C-terminal processing of the polypeptide encoded by cDNA. There are four potential N-linked glycosylation sites. Asn65, Asn72, Asn109, and Asn120. Sequence determination of isolated peptides suggested that Asn120 is glycosylated, Asn65 and Asn109 glycosylated in some molecules but not in others, and Asn72 not glycosylated. Amino acids at three positions, i.e. 142, 143, and 155, could not be detected during sequence analysis. Since the gene sequence codes for Ser, Thr, and Thr at these positions (Martin, F. H., Suggs, S. V., Langley, K. E., Lu, H. S., Ting, J., Okino, K. H., Morris, C. F., McNiece, I. K., Jacobsen, F. W., Mendiaz, E. A., Birkett, N. C., Smith, K. C., Johnson, M. J., Parker, V. P., Flores, J. C., Patel, A. C., Fisher, E. F., Erjavec, H. O., Herrera, C. J., Wypych, J., Sachdev, R. K., Pope, J. A., Leslie, I., Wen, D., Lin, C. W., Cupples, R. L., and Zsebo, K. M. (1990) Cell 63, 203-211), they could be sites of O-linked carbohydrate attachment. The four cysteines form two intramolecular disulfide bonds, Cys4-Cys89 and Cys43-Cys138.     

Crystal and molecular structure of the serine proteinase inhibitor CI-2 from barley seeds

Chymotrypsin inhibitor 2 (CI-2), a serine proteinase inhibitor from barley seeds, has been crystallized and its three-dimensional structure determined at 2.0-A resolution by the molecular replacement method. The structure has been refined by restrained-parameter least-squares methods to a crystallographic R factor (= sigma parallel Fo magnitude of-Fo parallel/sigma magnitude of Fo) o of 0.198. CI-2 is a member of the potato inhibitor 1 family. It lacks the characteristic stabilizing disulfide bonds of most other members of serine proteinase inhibitor families. The body of CI-2 shows few conformational changes between the free inhibitor and the previously reported structure of CI-2 in complex with subtilisin Novo [McPhalen, C.A., Svendsen, I., Jonassen, I., & James, M.N.G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7242-7246]. However, the reactive site loop has some significant conformational differences between the free inhibitor and its complexed form. The residues in this segment of polypeptide exhibit relatively large thermal motion parameters and some disorder in the uncomplexed form of the inhibitor. The reactive site bond is between Met-59I and Glu-60I in the consecutive sequential numbering of CI-2 (Met-60-Glu-61 according to the alignment of Svendsen et al. [Svendsen, I., Hejgaard, J., & Chavan, J.K. (1984) Carlsberg Res. Commun. 49, 493-502]). The network of hydrogen bonds and electrostatic interactions stabilizing the conformation of the reactive site loop is much less extensive in the free than in the complexed inhibitor.     

Chemical properties and amino acid composition of beta1-bungarotoxin from the venom of Bungarus multicinctus (Formosan banded krait)

beta-Bungarotoxin purified from the venom of Bungarus multicinctus (Formosan banded krait) contained no carbohydrate and behaved as a homogeneous protein on polyacrylamide gel electrophoresis at pH 4.1 and SDS-polyacrylamide gel electrophoresis without 2-mercaptoethanol treatment. Its molecular weight and isoelectric point were estimated to be about 21,000 by gel filtration and about 9.5 by isoelectric focusing, respectively. The toxin treated with the reducing agent was split into two polypeptide chains as revealed by SDS-polyacrylamide gel electrophoresis and their molecular weights were calculated to be about 13,000 and 7,000. The two polypeptide chains (the large one named the A chain and the small one the B chain) were isolated by gel filtration after reduction of disulfide bonds in the toxin followed by alkylation. The A chain contained 120 amino acid residues including 13 half-cystines and the B chain 60 residues including 7 half-cystines. The two chains were supposed to link by disulfide bond(s) in the intact toxin which contained no free sulfhydryl groups. The N-terminal residues of the A and B chains were asparagine and arginine and the C-terminal ones were glutamine and proline, respectively, in accordance with the results of the terminal analyses of the intact toxin.     

Roles of salt and conformation in the biological and physicochemical behavior of protegrin-1 and designed analogues: correlation of antimicrobial, hemolytic, and lipid bilayer-perturbing activities

Protegrins are short (16-18 residues) cationic peptides from porcine leukocytes that display potent, broad-spectrum antimicrobial activity. Protegrin-1 (PG-1), one of five natural homologues, adopts a rigid beta-hairpin structure that is stabilized by two disulfide bonds. We have previously employed the principles of beta-hairpin design to develop PG-1 variants that lack disulfide bonds but nevertheless display potent antimicrobial activity [Lai, J. R., Huck, B. R., Weisblum, B., and Gellman, S. H. (2002) Biochemistry 41, 12835-12842.]. The activity of these disulfide-free variants, however, is attenuated in the presence of salt, and the activity of PG-1 itself is not. Salt-induced inactivation of host-defense peptides, such as human defensins, is thought to be important in some pathological situations (e.g., cystic fibrosis), and the variation in salt-sensitivity among our PG-1 analogues offers a model system with which to explore the origins of these salt effects. We find that the variations in antimicrobial activity among our peptides are correlated with the folding propensities of these molecules and with the extent to which the peptides induce leakage of contents from synthetic liposomes. Comparable correlations were observed between folding and hemolytic activity. The extent to which added salt reduces antimicrobial activity parallels salt effects on vesicle perturbation, which suggests that the biological effects of high salt concentrations arise from modulation of peptide-membrane interactions.     

Intramolecular versus intermolecular disulfide bonds in prion proteins

Prion protein (PrP) is the major component of the partially protease-resistant aggregate that accumulates in mammals with transmissible spongiform encephalopathies. The two cysteines of the scrapie form, PrP(Sc), were found to be in their oxidized (i.e. disulfide) form (Turk, E., Teplow, D. B., Hood, L. E., and Prusiner, S. B. (1988) Eur. J. Biochem. 176, 21-30); however, uncertainty remains as to whether the disulfide bonds are intra- or intermolecular. It is demonstrated here that the monomers of PrP(Sc) are not linked by intermolecular disulfide bonds. Furthermore, evidence is provided that PrP(Sc) can induce the conversion of the oxidized, disulfide-intact form of the monomeric cellular prion protein to its protease-resistant form without the temporary breakage and subsequent re-formation of the disulfide bonds in cell-free reactions.     

Vesicle-binding properties of wild-type and cysteine mutant forms of alpha(1) domain of apolipoprotein B

Previous studies demonstrated that structural perturbation of the alpha(1) domain of apolipoprotein B (apoB) blocked the initiation of lipoprotein assembly. We explored the hypothesis that this domain may interact with the inner leaflet of the endoplasmic reticulum membrane in a manner that may nucleate microsomal triglyceride transfer protein-dependent lipid sequestration. ApoB-17 (amino-terminal 17% of apoB), which contains most of the alpha(1) domain, was expressed stably in rat hepatoma cells and recovered from medium in lipid-poor form. On incubation with phospholipid vesicles composed of 1-myristol-2-myristoyl-sn-glycero-3-phosphocholine or 1-palmitoyl-2-oleoyl-sn-gylycero-3-phosphocholine, apoB-17 underwent vesicle binding and was recovered in the d < 1.25 g/ml gradient fraction. To determine whether vesicle binding is disrupted by the same structural perturbations that block lipoprotein assembly in vivo, apoB-17 was subjected to partial and complete chemical reduction. Although normally a soluble peptide, mild reduction of apoB-17 caused its precipitation, suggesting that hydrophobic, solvent-inaccessible domains within the alpha(1) domain of apoB are stabilized by intramolecular disulfide bonds. In contrast to apoB-17 chemically reduced in vitro, forms of apoB-17 bearing pairwise cysteine-to-serine substitutions were recovered in soluble form from transiently transfected COS-1 cell extracts. Although individual disruption of disulfide bond 2 or 4 in apoB-28 and apoB-50 was previously shown to block lipoprotein assembly in vivo, these alterations had no impact on the ability of apoB-17 to bind to phospholipid vesicles in vitro or on its capacity to form recombinant lipoprotein particles. These results suggest that while the vesicle/lipid-binding property of the alpha(1) domain may reflect an essential role required for the initiation of lipoprotein formation, some other aspect of alpha(1) domain function is perturbed by disruption of native disulfide bonds. -- DeLozier, J. A., J. S. Parks, and G. S. Shelness. Vesicle-binding properties of wild-type and cysteine mutant forms of alpha(1) domain of apolipoprotein B. J. Lipid Res. 2001. 42: 399--406.     

Human alpha 1,3/4 fucosyltransferases. Characterization of highly conserved cysteine residues and N-linked glycosylation sites

Human alpha1,3 fucosyltransferases (FucTs) contain four highly conserved cysteine (Cys) residues, in addition to a free Cys residue that lies near the binding site for GDP-fucose (Holmes, E. H., Xu, Z. , Sherwood, A. L., and Macher, B. A. (1995) J. Biol. Chem. 270, 8145-8151). The participation of the highly conserved Cys residues in disulfide bonds and their functional significance were characterized by mass spectrometry (MS) analyses and site-directed mutagenesis, respectively. Among the human FucTs is a subset of enzymes (FucT III, V, and VI) having highly homologous sequences, especially in the catalytic domain, and Cys residues in FucT III and V were characterized. The amino acid sequence of FucT III was characterized. Peptides containing the four conserved Cys residues were detected after reduction and alkylation, and found to be involved in disulfide bonds. The disulfide bond pattern was characterized by multiple stage MS analysis and the use of Glu-C protease and MS/MS analysis. Disulfide bonds in FucT III occur between Cys residues (Cys(81) to Cys(338) and Cys(91) to Cys(341)) at the N and C termini of the catalytic domain, bringing these ends close together in space. Mutagenesis of highly conserved Cys residues to Ser in FucT V resulted in proteins lacking enzymatic activity. Three of the four mutants have molecular weights similar to wild type enzyme and maintained an ability to bind GDP, whereas the other (Cys(104)) produced a series of lower molecular weight bands when characterized by Western blot analysis, and did not bind GDP. FucTs have highly conserved, potential N-linked sites, and our mass spectrometry analyses demonstrated that both N-linked sites are modified with oligosaccharides.     

Structure of human lysosomal membrane glycoprotein 1. Assignment of disulfide bonds and visualization of its domain arrangement

The amino acid sequence of one of the major lysosomal membrane glycoproteins, lysosome-associated membrane protein 1 (lamp-1), was deduced from its cDNA sequence (Fukuda, M., Viitala, J., Matteson, J., and Carlsson, S. R. (1988) J. Biol. Chem. 263, 18920-18928). This amino acid sequence suggests that lamp-1 contains a hinge-like structure and could form disulfide bridges that are observed in the immunoglobulin superfamily. To test this possibility, we have determined the positions of the disulfide bridges by isolating and sequencing cystine-containing peptides which contain disulfide bridges. The results indicate that disulfide arrangement of lamp-1 is different from that of immunoglobulins. Each molecule contains, in total, four loops formed by disulfide bonds, and each loop contains 36-39 amino acid residues. However, none of the disulfide bonds connects two domains that are separated by a hinge-like structure. The results indicate that the hinge region has no ordered structure, and the relative positions of the two domains can be altered in space. Examination of the ultrastructure of lamp-1 by electron microscopy showed that the hinge-like structure actually functions as a hinge. These results indicate that the lamp-1 molecule represents a novel family of glycoproteins with unique structural properties.