All three potential N-glycosylation sites of the dog kidney (Na+ + K+)-ATPase beta-subunit contain oligosaccharide

The beta-subunit of dog kidney (Na+ + K+)-ATPase is a sialoglycoprotein and contains three potential N-glycosylation sites. In this study, the oligosaccharide chains of purified dog kidney beta-subunit were labeled with tritium by oxidation with sodium periodate or galactose oxidase followed by NaB3H4 reduction. The beta-subunit was extensively digested by trypsin and the radioactive peptides were purified by HPLC. The enzyme, glycopeptidase A, which catalyzes the removal of N-linked oligosaccharide chains and the conversion of the glycosylated Asn residue to Asp, was used to demonstrate that a number of purified beta-subunit tryptic peptides were glycosylated. Amino-acid analysis of these beta-subunit peptides following glycopeptidase-A treatment revealed the expected Asn to Asp conversion for Asn-157, Asn-192 and Asn-264, demonstrating that all three potential N-glycosylation sites of the dog kidney beta-subunit are glycosylated. In addition, amino-acid sequence data suggest that a disulfide bond exists between Cys-158 and Cys-174.     

Characterization of a soluble form of human CD4. Peptide analyses confirm the expected amino acid sequence, identify glycosylation sites and demonstrate the presence of three disulfide bonds

CD4 is a glycoprotein that is expressed on the surface of a variety of cells of the immune system and is believed to participate in the interactions of these cells with antigen-presenting cells bearing the class II major histocompatibility (MHC) antigens. CD4 also acts as the receptor for the human immunodeficiency virus (HIV) by binding to the viral glycoprotein gp120. Recombinant soluble CD4 (rCD4) is a truncated form of human CD4 that is secreted from transfected Chinese hamster ovary cells. This 368-amino-acid glycoprotein contains two potential sites of N-linked glycosylation (Asn-271 and Asn-300) and six cysteine residues. Amino-terminal sequence analysis demonstrated that the sequence begins at the third residue of the polypeptide originally predicted from the cDNA analysis [Maddon, P.J. et al. (1985) Cell 42, 93-104]. The rest of the primary sequence was confirmed by analysis of peptides purified by reversed-phase HPLC after digestion of S-carboxymethylated rCD4 with trypsin. Anhydrotrypsin affinity chromatography of trypsin-digested rCD4 confirmed that the carboxy-terminus of the protein was Pro-368. Enzymatic digestion of non-reduced rCD4 generated disulfide-bonded fragments that demonstrated the presence of disulfide bonds between Cys-16 and Cys-84, Cys-130 and Cys-159, and between Cys-303 and Cys-345. The constituent monosaccharides of the carbohydrate structures of rCD4 were found to be fucose, mannose, galactose, N-acetylglucosamine and N-acetylneuraminic acid. Characterization of the tryptic map of rCD4 after treatment with peptide: N-glycosidase F demonstrated that both potential N-glycosylation sites are utilized. The tryptic map of rCD4 treated with endo-beta-N-acetylglucosamine H demonstrated that only complex-type oligosaccharides are attached to Asn-271, while Asn-300 has high-mannose or hybrid structures attached in addition to complex-type oligosaccharides. Glucosamine was observed only in glycopeptides that contain Asn-300 or Asn-271 while no galactosamine was observed. This suggests that rCD4 contains no O-linked oligosaccharides.     

Structure of recombinant human interleukin 5 produced by Chinese hamster ovary cells

The complete peptide map of purified recombinant human interleukin 5 (rhIL-5) was determined to verify its primary structure, glycosylation sites, and disulfide bonding structure. Each peptide fragment generated by Achromobacter protease I (API) digestion was purified and characterized by amino acid analysis and amino acid sequence analysis. After digestion with API, we could identify all the peptides which were expected from human IL-5 cDNA sequence. The analyses of sulfhydryl content in rhIL-5 molecule and disulfide-containing peptide obtained from API digestion indicated that active form of rhIL-5 existed as an antiparallel dimer linked by two pairs of Cys-44 and Cys-86. In addition, we concluded that Thr-3 and Asn-28 were glycosylated. The results indicate that primary structure of rhIL-5 is highly homogeneous and observed heterogeneity is due to the difference in the content of carbohydrate.     

Purification, characterization, and the complete amino acid sequence of porcine pancreatic deoxyribonuclease

Porcine pancreatic DNase has been purified to homogeneity. The polypeptide exhibits a single band of Mr = 34,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is a glycoprotein containing glucosamine. The results of end group analyses show leucine at the NH2 terminus and alanine at the COOH terminus. The enzymatic properties of the purified porcine DNase are very similar to those of bovine and ovine DNases. The sequence data on the tryptic and chymotryptic peptides derived from CNBr fragments of porcine DNase, along with the results of automated Edman degradation of the intact polypeptide and of the two largest CNBr fragments, indicate the complete amino acid sequence of porcine DNase to be as follows:L-R- I-A-F-N-I-R-T-F-G-E-T-K-M-S-N-A-T-S-N-Y-I-V-R-I-L-S-R-Y-D-I-A-L-I-Q- E-V-R-D-S-H-L-T-A-V-G-K-L-L-N-E-L-N-Q-D-D-P-N-N-Y-H-H-V-V-S-E-P-L-G-R- S-T-Y-K-E-R-Y-L-F-V-F-R-P-N-Q-V-S-V-L-D-S-Y-L-Y-D-D-G-C-E-P-C-G-N-D-T- F-N-R-E-P-S-V-V-K-F-S-S-P-F-T-Q-V-K-E-F-A-I-V-P-L-H-A-A-P-S-D-A-A-A-E- I-N-S-L-Y-D-V-Y-L-N-V-R-Q-K-W-D-L-Q-D-I-M-L-M-G-D-F-N-A-G-C-S-Y-V-T- T-S-H-W-S-S-I-R-L-R-E-S-P-P-F-Q-W-L-I-P-D-T-A-D-T-T-V-S-S-H-T-C-A-Y- D-R-I-V-V-A-G-P-L-L-Q-R-A-V-V-P-D-S-A-A-P-F-D-F-Q-A-A-F-G-L-S-Q-E-T- A-L-A-I-S-D-H-Y-P-V-E-V-T-L-K-R-A. The polypeptide consists of 262 amino acid residues. One of the two disulfide loops links Cys-101 and Cys-104 and the other Cys-173 and Cys-209. Two carbohydrate side chains are attached at Asn-18 and Asn-106.     

Determination of disulfide bond assignment of human vitamin K-dependent gamma-glutamyl carboxylase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Vitamin K-dependent gamma-glutamyl carboxylase is a 758 amino acid integral membrane glycoprotein that catalyzes the post-translational conversion of certain protein glutamate residues to gamma-carboxyglutamate. Carboxylase has ten cysteine residues, but their form (sulfhydryl or disulfide) is largely unknown. Pudota et al. in Pudota, B. N., Miyagi, M., Hallgren, K. W., West, K. A., Crabb, J. W., Misono, K. S., and Berkner, K. L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 13033-13038 reported that Cys-99 and Cys-450 are the carboxylase active site residues. We determined the form of all cysteines in carboxylase using in-gel protease digestion and matrix-assisted laser desorption/ionization mass spectrometry. The spectrum of non-reduced, trypsin-digested carboxylase revealed a peak at m/z 1991.9. Only this peak disappeared in the spectrum of the reduced sample. This peak's m/z is consistent with the mass of peptide 92-100 (Cys-99) disulfide-linked with peptide 446-453 (Cys-450). To confirm its identity, the m/z 1991.9 peak was isolated by a timed ion selector as the precursor ion for further MS analysis. The fragmentation pattern exhibited two groups of triplet ions characteristic of the symmetric and asymmetric cleavage of disulfide-linked tryptic peptides containing Cys-99 and Cys-450. Mutation of either Cys-99 or Cys-450 caused loss of enzymatic activity. We created a carboxylase variant with both C598A and C700A, leaving Cys-450 as the only remaining cysteine residue in the 60-kDa fragment created by limited trypsin digestion. Analysis of this fully active mutant enzyme showed a 30- and the 60-kDa fragment were joined under non-reducing conditions, thus confirming Cys-450 participates in a disulfide bond. Our results indicate that Cys-99 and Cys-450 form the only disulfide bond in carboxylase.     

Determination of disulfide array and subunit structure of taste-modifying protein, miraculin

The taste-modifying protein, miraculin (Theerasilp, S. et al. (1989) J. Biol. Chem. 264, 6655-6659) has seven cysteine residues in a molecule composed of 191 amino acid residues. The formation of three intrachain disulfide bridges at Cys-47-Cys-92, Cys-148-Cys-159 and Cys-152-Cys-155 and one interchain disulfide bridge at Cys-138 was determined by amino acid sequencing and composition analysis of cystine-containing peptides isolated by HPLC. The presence of an interchain disulfide bridge was also supported by the fact that the cystine peptide containing Cys-138 showed a negative color test for the free sulfhydryl group and a positive test after reduction with dithiothreitol. The molecular mass of non-reduced miraculin (43 kDa) in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was nearly twice the calculated molecular mass based on the amino acid sequence and the carbohydrate content of reduced miraculin (25 kDa). The molecular mass of native miraculin determined by low-angle laser light scattering was 90 kDa. Application of a crude extract of miraculin to a Sephadex G-75 column indicated that the taste-modifying activity appears at 52 kDa. It was concluded that native miraculin in pure form is a tetramer of the 25 kDa-peptide and native miraculin in crude state or denatured, non-reduced miraculin in pure form is a dimer of the peptide. Both tetramer miraculin and native dimer miraculin in crude state had the taste-modifying activity.     

Determination of disulfide bond assignments and N-glycosylation sites of the human gastrointestinal carcinoma antigen GA733-2 (CO17-1A, EGP, KS1-4, KSA, and Ep-CAM)

The GA733-2 antigen is a cell surface glycoprotein highly expressed on most human gastrointestinal carcinoma and at a lower level on most normal epithelia. It is an unusual cell-cell adhesion protein that does not exhibit any obvious relationship to the four known classes of adhesion molecules. In this study, the disulfide-bonding pattern of the GA733-2 antigen was determined using matrix-assisted laser desorption/ionization mass spectrometry and N-terminal sequencing of purified tryptic peptides treated with 2-[2'-nitrophenylsulfonyl]-3-methyl-3-bromoindolenine or partially reduced and alkylated. Numbering GA733-2 cysteines sequentially from the N terminus, the first three disulfide linkages are Cys1-Cys4, Cys2-Cys6, and Cys3-Cys5, which is a novel pattern for a cysteine-rich domain instead of the expected epidermal growth factor-like disulfide structure. The next three disulfide linkages are Cys7-Cys8, Cys9-Cys10, and Cys11-Cys12, consistent with the recently determined disulfide pattern of the thyroglobulin type 1A domain of insulin-like growth factor-binding proteins 1 and 6. Analysis of glycosylation sites showed that GA733-2 antigen contained N-linked carbohydrate but that no O-linked carbohydrate groups were detected. Of the three potential N-linked glycosylation sites, Asn175 was not glycosylated, whereas Asn88 was completely glycosylated, and Asn51 was partially glycosylated. These data show that the extracellular domain of the GA733-2 antigen consists of three distinct domains; a novel cysteine-rich N-terminal domain (GA733 type 1 motif), a cysteine-rich thyroglobulin type 1A domain (GA733 type 2 motif), and a unique nonglycosylated domain without cysteines (GA733 type 3 motif).     

Mass spectrometric determination of glycosylation sites and oligosaccharide composition of insect-expressed mouse interleukin-3

The primary structure of Baculovirus-expressed mouse interleukin-3 produced in infected Bombyx mori larvae was characterized by liquid secondary ion mass spectrometry and ²⁵²Cf-plasma desorption mass spectrometry in combination with selected protein microchemical reactions. Interleukin-3 was found to consist of at least two glycoprotein species of ca. 17 000 dalton. Characterization of tryptic and S. aureus V8 protease peptides by Edman degradation combined with plasma desorption mass spectrometry showed that two N-glycosylation sites, Asn-16 and Asn-86, were present. N-Glycan residues were shown by liquid secondary ion mass spectrometry and high-performance liquid chromatography to consist of mannose, fucose, and glucosamine. The presence of galactosamine indicated that O-glycosylated residues were present, in addition to the N-glycosylated residues. Glucose was also present, which indicated incomplete processing of the insect-expressed N-linked oligosaccharides.     

Rat brain Thy-1 glycoprotein. The amino acid sequence, disulphide bonds and an unusual hydrophobic region.

The full sequence of the Thy-1 membrane glycoprotein of rat brain is reported. The sequence was determined from tryptic and V-8 proteinase peptides and consisted of 111 amino acids. The amino terminus was blocked and consisted of a pyroglutamic acid residue. The molecule contained two disulphide bonds, namely Cys-9--Cys-111 and Cys-19--Cys-85. Three N-linked amino sugars were located at Asn-23, Asn-74 and Asn-98. In each case the sequence on the C-terminal side of the attachment point was Asn-Xaa-Thr as would be expected for N-linkage. The C-terminal peptides were unusual, in that they were either obtained in a highly aggregated form, or could only be purified after binding to Brij 96 micelles. Thus they appeared to have hydrophobic properties, yet did not contain any extended sequence of hydrophobic amino acids. Other unusual features of the C-terminal peptides were the presence of unidentified ninhydrin-positive material and of glucosamine and galactosamine. The C-terminal residue has not been directly identified but Cys-111 is the last conventional amino acid. It is suggested that the hydrophobic properties of the C-terminal peptides may be due to the linkage of lipid. The sequence of the Thy-1 glycoprotein showed homologies with immunoglobulin domains. This relationship is examined in detail in the paper following [Cohen et al. (1981) Biochem. J. 193, 000--000].     

Studies on the structure of rabbit muscle aldolase: Amino acid sequence of cysteine-containing peptides

Five cysteine-containing peptides have been isolated in nearly stoichemometric yields from the tryptic digests of the NH₂^? and COOH-terminal BrCN peptides of rabhit muscle aldolase and their sequence determined. Peptides NS1, NS2, and NS3, from the NH₂-terminal part of the enzyme have the following sequences: NS1, Val-Asp-Pro-Cys-Ile-Gly-Gly-Val-Ile-Leu-Phe-His-Glu-Thr-Leu-Tyr-Gln-Lys; NS2, Cys-Val-Leu-Lys; NS3, Cys-Ala-Glu-Tyr-Lys. The two peptides isolated from the COOH-terminal region are: CS1, Ala-Leu-Ala-Asn-Ser-Leu-Ala-Cys-Gln-Gly-Lys and CS2, Cys-Pro-Leu-Leu-Trp-Pro-Lys-Ala-Leu-Thr-Phe-Ser-Tyr-Gly-Arg. The Lys-Ala bond in peptide CS2 was found to be resistant to tryptic hydrolysis. The results provide the basis for assigning the positions of cysteine residues in the polypeptide chain. Cys-72 in peptide NS1 and Cys-336 in peptide CS1 are the residues that form a disulfide bridge when the enzyme is inactivated by oxidation with an o-phenanthroline-Cu²⁺ complex; Cys-287 in peptide CS2 in one of the two exposed residues, while Cys-134 and Cys-149 in peptides NS2 and NS3, respectively, are buried in the native enzyme. All of eight cysteine-containing peptides of rabbit muscle aldolase have now been sequenced, and structural homology of the α and β subunits extended to these regions.     

Glycosylations and truncations of functional cereal phytases expressed and secreted by Pichia pastoris documented by mass spectrometry

Cereal purple acid phosphatase-type phytases, PAPhy, play an essential role in making phosphate accessible to mammalian digestion and reducing the environmental impact of manure. Studying the potential of PAPhy requires easy access to the enzymes. For that purpose wheat and barley isophytases have been expressed in Pichia pastoris from constructs encoding the alpha-mating factor at the N-termini and a His? tag before the stop codon in all constructs. A protein chemical study of a C-terminally truncated recombinant wheat phytase, r-TaPAPhy_b2, was carried out to clarifying the posttranslational processing of proteins secreted from P. pastoris. Extensive mass spectrometric sequencing of tryptic, chymotryptic and AspN derived peptides of both the native and endoH deglycosylated forms showed: (i) All mating factor derived sequence had been removed and further unspecific proteolysis left highly heterogeneous N-terminal variant forms of r-TaPAPhy; (ii) The His? tag had been retained or slightly truncated; (iii) All seven potential N-glycan sites were glycosylated except for two sites which were partially glycosylated by ca. 90% and 30%; (iv) Among the nine cysteine residues of this phytase, the most N-terminal residue is free, whereas the remaining eight appear to be disulfide bonded. It is noteworthy that already the first step in ESI-MS/MS sequencing had fragmented the hyper glycosylated peptides into free Z, Y and X mass spectrometric glycan fragments attached to the peptide.     

Manduca sexta hemolymph protease-1, activated by an unconventional non-proteolytic mechanism,mediates immune responses

Tissue damage or pathogen invasion triggers the auto-proteolysis of an initiating serine protease (SP), rapidly leading to sequential cleavage activation of other cascade members to set off innate immune responses in insects. Recently, we presented evidence that Manduca sexta hemolymph protease-1 zymogen (proHP1) is a member of the SP system in this species, and may activate proHP6. HP6 stimulates melanization and induces antimicrobial peptide synthesis. Here we report that proHP1 adopts an active conformation (*) to carry out its function, without a requirement for proteolytic activation. Affinity chromatography using HP1 antibodies isolated from induced hemolymph the 48 kDa proHP1 and also a 90 kDa band (detected by SDS-PAGE under reducing conditions) containing proHP1 and several serpins, as revealed by mass spectrometric analysis. Identification of tryptic peptides from these 90 kDa complexes included peptides from the amino-terminal regulatory part of proHP1, indicating that proHP1* was not cleaved, and that it had formed a complex with the serpins. As suicide inhibitors, serpins form SDS-stable, acyl-complexes when they are attacked by active proteases, indicating that proHP1* was catalytically active. Detection of M. sexta serpin-1, 4, 9, 13 and smaller amounts of serpin-3, 5, 6 in the complexes suggests that it is regulated by multiple serpins in hemolymph. We produced site-directed mutants of proHP1b for cleavage by bovine blood coagulation factor Xa at the designed proteolytic activation site, to generate a form of proHP1b that could be activated by Factor Xa. However, proHP1b cut by Factor Xa failed to activate proHP6 and, via HP6, proHP8 or proPAP1. This negative result is consistent with the suggestion that proHP1* is a physiological mediator of immune responses. Further research is needed to investigate the conformational change that results in conversion of proHP1 to active proHP1*.     

Use of the silkworm, Bombyx mori, and an insect baculovirus vector for high-level expression and secretion of biologically active mouse interleukin-3

Using the virus vector derived from a baculovirus of Bombyx mori (Bm), we constructed an infectious recombinant virus carrying the mouse interleukin-3 (IL-3) cDNA placed downstream from the polyhedrin promoter. Silkworms infected in vivo with recombinant virus or the silkworm-derived BmN cell line infected in vitro secreted large amounts of IL-3 into hemolymph or culture medium, respectively. On a per volume basis, about 20-fold more activity was found in the culture supernatants of the infected BmN cells and 10000-fold more activity was detected in the hemolymph as compared to supernatants obtained from COS7 monkey cells transfected with plasmid pcD-IL3 using the SV40 early promoter [Yokota et al., Proc. Natl. Acad. Sci. USA 81 (1984) 1070-1074]. Three distinct species of Il-3 of molecular masses, 18, 20 and 22 kDa were produced and all were converted to a 15-kDa protein by N-glycanase digestion, indicating that silkworm cells glycosylated IL-3. The N-terminal amino acid sequences of the IL-3 purified from tissue culture medium and hemolymph were identical to that of mammalian-derived IL-3, showing that silkworm cells recognized the mammalian signal sequence and cleaved it at the correct position. The purified silkworm-produced IL-3 had biological activities indistinguishable from IL-3 produced by mammalian cells as assessed by mast-cell proliferation assays, colony-formation assays using mouse bone marrow cells, and by receptor-binding assays using [125I]IL-3.     

Disulfide bonds of herpes simplex virus type 2 glycoprotein gB.

Glycoprotein B (gB) is the most highly conserved envelope glycoprotein of herpesviruses. The gB protein is required for virus infectivity and cell penetration. Recombinant forms of gB being used for the development of subunit vaccines are able to induce virus-neutralizing antibodies and protective efficacy in animal models. To gain structural information about the protein, we have determined the location of the disulfide bonds of a 696-amino-acid residue truncated, recombinant form of herpes simplex virus type 2 glycoprotein gB (HSV gB2t) produced by expression in Chinese hamster ovary cells. The purified protein, which contains virtually the entire extracellular domain of herpes simplex virus type 2 gB, was digested with trypsin under nonreducing conditions, and peptides were isolated by reversed-phase high-performance liquid chromatography (HPLC). The peptides were characterized by using mass spectrometry and amino acid sequence analysis. The conditions of cleavage (4 M urea, pH 7) induced partial carbamylation of the N termini of the peptides, and each disulfide peptide was found with two or three different HPLC retention times (peptides with and without carbamylation of either one or both N termini). The 10 cysteines of the molecule were found to be involved in disulfide bridges. These bonds were located between Cys-89 (C1) and Cys-548 (C8), Cys-106 (C2) and Cys-504 (C7), Cys-180 (C3) and Cys-244 (C4), Cys-337 (C5) and Cys-385 (C6), and Cys-571 (C9) and Cys-608 (C10). These disulfide bonds are anticipated to be similar in the corresponding gBs from other herpesviruses because the 10 cysteines listed above are always conserved in the corresponding protein sequences.     

The site of linkage of a retinoid affinity label to plasma retinol-binding protein

The retinoid affinity label 11[³H]--ionylidene ethylbromoacetate (IEBA) was covalently bound to plasma retinol-binding protein (RBP) and studies were conducted to identify the region of the protein molecule that contained the linkage between the IEBA ligand and RBP. Cleavage by trypsin and cyanogen bromide of the labeled protein followed by high-performance liquid chromatography (HPLC) separation of peptides and identification of radioactive peaks by amino acid analysis points to attachment of the ligand on tryptic peptides T(1+2) (containing residues 1–5) and T(21) (residues 156–163). These two peptides in the native protein molecule are connected by a disulfide bond between Cys-4 and Cys-160. To confirm the site of attachment of the radioactive ligand, unreduced IEBA-RBP with the disulfide bonds intact was treated first with cyanogen bromide and then with trypsin. Separation of the tryptic peptides by HPLC yielded one main peak of radioactivity containing both peptides T(1+2) and T(21), presumably connected by a disulfide bond. Taken together, these results indicated that the sites of attachment of IEBA to RBP are located within the region of the RBP molecule close to the Cys-4–Cys-160 bond, and specifically within the region comprised of amino acid residues 1–5 and 156–163.     

Acetylcholine receptor binding site contains a disulfide cross-link between adjacent half-cystinyl residues

A conserved feature of all nicotinic receptors is the presence of a readily reducible disulfide bond adjacent to the acetylcholine binding site. Previously we showed that in intact receptor from Torpedo californica electric tissue reduction of this disulfide followed by affinity alkylation with 4-(N-maleimido)benzyltri[3H] methylammonium iodide specifically and uniquely labels the alpha subunit residues Cys-192 and Cys-193. To identify all of the half-cystinyl residues contributing to the binding site disulfide(s), we have now reduced receptor under mild conditions and alkylated with a mixture of 4-(N-maleimido)benzyltri[3H]methylammonium iodide and N-[1-14C]ethylmaleimide and find that Cys-192 and Cys-193 are labeled exclusively. Furthermore, from unreduced receptor we have isolated two cyanogen bromide peptides of alpha, one containing Cys-192 and Cys-193, and the other containing Cys-128 and Cys-142 (which are the other potential contributors to the binding site disulfide(s]. These isolated peptides incorporate iodo[1-14C]acetamide only following reduction by dithiothreitol. Our results demonstrate that: 1) the binding site disulfide is between Cys-192 and Cys-193; 2) Cys-128 is disulfide-cross-linked to Cys-142; and 3) under conditions that reduce Cys-192 and Cys-193 completely, Cys-128 and Cys-142 remain cross-linked. At the acetylcholine binding site, agonists induce a local conformational change that stabilizes the binding site disulfide against reduction. We suggest that a transition between two stable conformations of the vicinal disulfide, both involving a nonplanar cis peptide bond between Cys-192 and Cys-193, is associated with receptor activation by agonists.     

Disulfide scrambling of interleukin-2: HPLC resolution of the three possible isomers

Native interleukin-2 (IL-2) contains three cysteines; two exist in a disulfide bridge (Cys-58 and Cys-105) and the third Cys-125 is a free sulfhydryl. In the presence of 6 M guanidine hydrochloride at alkaline pH, IL-2 is converted into three isomers. Each isomer represents one of the three possible disulfide-linked forms that can be generated from three cysteines. These three isomers were resolved on a C4 reverse-phase HPLC system. The identity of each of the three forms was determined by carboxymethylation of the free cysteines in each isomer with [3H]iodoacetic acid followed by determination of the labelled cysteines by tryptic peptide mapping. Tryptic peptide mapping of the more predominant of the two scrambled peaks showed it to be the Cys-105-S-S-Cys-125 linked form of IL-2. A Ser-125 construction of IL-2, which lacks a free cysteine, did not scramble under these conditions. These experiments demonstrate the utility of reverse-phase HPLC in studies of protein folding and disulfide bond structure.     

The structure of jack bean urease. The complete amino acid sequence, limited proteolysis and reactive cysteine residues

The amino acid sequence of jack bean urease has been determined. The protein consists of a single kind of polypeptide chain containing 840 amino acid residues. The subunit relative molecular mass calculated from the sequence is 90,770, indicating that urease is composed of six subunits. Out of 25 histidine residues in urease, 13 were crowded in the region between residues 479 and 607, suggesting that this region may contain the nickel-binding site. Limited tryptic digestion cleaved urease at two sites, Lys-128 and Lys-662. Proteolytic products were not dissociated and retained full enzymatic activity. Five tryptic peptides containing the reactive cysteine residues were isolated and characterized with the aid of sulfhydryl-specific reagents, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine and N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide. The reactive cysteine residues were located at positions 59, 207, 592, 663, and 824. The possibility that Cys-59, Cys-207, Cys-663, and Cys-824 are involved in the urease activity of the enzyme has been eliminated. Cys-592, which is essential for enzymatic activity, is located in the above-mentioned histidine-rich region.     

Differences in glycosylation pattern of human secretory ribonucleases.

The major secretory ribonuclease (RNase) of human urine (RNase HUA) was isolated and sequenced by automatic Edman degradation and analysis of peptides and glycopeptides. The isolated enzyme was shown to be free of other urine RNase activities by SDS/polyacrylamide-gel electrophoresis and activity staining. It is a glycoprotein 128 amino acids long, differing from human pancreatic RNase in the presence of an additional threonine residue at the C-terminus. It differs from the pancreatic enzyme in its glycosylation pattern as well, and contains about 45 sugar residues. Each of the three Asn-Xaa-Ser/Thr sequences (Asn-34, Asn-76, Asn-88) is glycosylated with a complex-type oligosaccharide chain. Glycosylation at Asn-88 has not been observed previously in mammalian secretory RNases. Preliminary sequence data on the major RNase of human seminal plasma have revealed no difference between it and the major urinary enzyme; their similarities include the presence of threonine at the C-terminus. The glycosylation pattern of human seminal RNase is very similar to that of the pancreatic enzyme. The structural differences between the secretory RNases from human pancreas, urine and seminal plasma must originate from organ-specific post-translational modifications of the one primary gene product. Detailed characterization of peptides and the results of gel filtration of tryptic and tryptic/chymotryptic digests of performic acid-oxidized RNase have been deposited as Supplementary Publication SUP 50146 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1988) 249, 5.     

Use of recombinant DNA derived human relaxin to probe the structure of the native protein

This report describes the physical, chemical, and biological characterization of recombinant human relaxin (rhRlx) used as a probe to establish the disulfide pairing in native human relaxin. This strategy is necessary since native human relaxin is only available in the nanogram range. The relaxin molecule is composed of two nonidentical peptide chains, an A-chain 24 amino acids in length and a B-chain of 29 amino acids, linked by two disulfide bridges with an additional disulfide linkage in the A-chain. Native relaxin isolated from human corpora lutea was compared to rhRlx by reversed-phase chromatography, partial sequence analysis, mass spectroscopy, and bioassay. The potency of rhRlx was established by its ability to stimulate cAMP from primary human uterine endometrial cells. Native relaxin isolated from human corpora lutea was equipotent to chemically synthesized relaxin, which in turn was equipotent to rhRlx. A tryptic map was developed for rhRlx to confirm the complete amino acid sequence and assignment of the disulfide bonds. The three disulfide bonds (CysA10-CysA15, CysA11-CysB11, and CysA24-CysB23) were assigned by mass spectrometric analysis of the tryptic peptides and by comparison to chemically synthesized peptides disulfide linked in the two most probable configurations. In addition, the observed amino acid composition and sequence of rhRlx was in agreement with that predicted from the cDNA sequence with the exception that the A-chain amino terminal was pyroglutamic acid. The migration of rhRlx upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis was consistent with a monomeric structure, and the identity of the band was demonstrated by immunoblotting.