Primary structure of a human IgA1 immunoglobulin. II. Isolation, composition, and amino acid sequence of the tryptic peptides of the whole alpha1 chain and its cyanogen bromide fragments.

As part of the strategy for determining the covalent structure of a human IgA1 molecule (Bur), a tryptic digest was prepared of the reduced and carboxymethylated alpha1 heavy chain. In addition to the main experiment, tryptic peptides were prepared from the succinylated aminoethylated alpha1 chain and from fragments obtained by CNBr scission of the alpha1 chain. Complete recovery of the peptides was impeded by the large size of some of the tryptic peptides and of the principal CNBr fragment, and difficulty in separating other glycopeptides. Twenty-eight tryptic peptides of the reduced and carboxymethylated alpha1 chain were purified and sequenced, accounting for more than 300 residues. Additional information was obtained by sequence analysis of trypudies described in this series of papers contributed to the complete sequence analysis of the alpha1 chain.     

Primary structure of a human IgA1 immunoglobulin. IV. Streptococcal IgA1 protease, digestion, Fab and Fc fragments, and the complete amino acid sequence of the alpha 1 heavy chain.

In order to establish the complete amino acid sequence of the human IgA alpha1 chain Bur, IgA1 protease from Streptococcus sanguis was employed to generate Fabalpha and Fcalpha fragments in the final stage of this investigation. Cyanogen bromide cleavage of the Fabalpha fragment followed by reduction and aminoethylation produced the Fd' fragment (residues 84 to 227); this contains part of the variable region (VR), the whole first constant domain (Calpha1), and part of the hinge region of this heavy chain. The tryptic peptides of the Fd' fragment were isolated, characterized, and sequenced. The results together with the data in the preceding papers on chymotryptic, tryptic, and thermolysin peptides permitted the complete amino acid sequence of the human IgA alpha1 chain to be established.     

Primary structure of a human IgA1 immunoglobulin. I. Isolation, composition, and amino acid sequence of the chymotryptic peptides.

As the initial phase of the determination of the complete covalent structure of a human immunoglobulin A, 52 chymotryptic peptides, ranging in length from 2 to 37 residues, were isolated and characterized from the reduced and carboxymethylated alpha1 heavy chain of the myeloma IgA protein Bur. The peptides were subjected to sequence analysis by the dansylation technique, manual and automatic Edman degradation, and carboxypeptidase digestion. The results, in conjunction with the data on the tryptic and thermolysin peptides and the cyanogen bromide fragments reported in the accompanying papers, established the complete primary structure of a human IgA chain.     

Primary structure of the calcium ion-transporting adenosine triphosphatase from rabbit skeletal sarcoplasmic reticulum. Some peptic, thermolytic, tryptic and staphylococcal-proteinase peptides.

The soluble peptides from the peptic digest of the reduced S-carboxymethylated 3-carboxypropionylated adenosine triphosphatase protein have been isolated and most of their structures have been determined. About 397 residues of the protein were represented in these peptides. The reduced S-carboxymethylated protein was digested with thermolysin, and peptides containing arginine or carboxymethylcysteine were isolated and characterized. Some peptides isolated from tryptic and staphylococcal-proteinase digests of the protein are described. The information contained within the structures of these peptides has been used to reconstruct long stretches of the sequence of the ATPase protein that constitute most of the protein structure external to the lipid bilayer (Allen, Trinnaman and Green (1980) Biochem. J. 187, 591-616). The details of some of the chromatographic steps used in the isolation of the peptides and the properties of the peptides are contained in Supplementary Publication SUP 50104 (45 pages), which has been deposited with 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. (1978) 169, 5.     

Covalent structure of collagen: amino acid sequence of three cyanogen bromide-derived peptides from human alpha 1(V) collagen chain

Type V collagen was prepared from human amnionic/chorionic membranes and separated into alpha 1(V) and alpha 2(V) polypeptide chains. The alpha 1(V) chain was digested with cyanogen bromide and nine peptides were obtained and purified. Three of the peptides, alpha 1(V)CB1, CB4, and CB7 having molecular weights of 5000, 8000, and 6000, respectively, were further analyzed by amino acid sequence analysis and thermolytic or tryptic digestions. CB1 contained 54 amino acids and identification of its complete sequence was aided by thermolysin digestion and isolation of two peptides, Th1 and Th2. CB4 contained 81 amino acids and sequence analysis of intact CB4 and five tryptic peptides provided us with its complete amino acid sequence. The peptide CB7 contained 67 amino acids and was cleaved into four tryptic peptides that were used for complete sequence analysis. The above results represent the first available covalent structure information on the alpha 1(V) collagen chain. These data enabled us to establish the location of these peptides within the helical structure of other collagen chains. CB4 was homologous to residues 66-145 in the collagen chain while CB1 represented residues 146-200 and CB7 was homologous with residues 201-269. This alignment was facilitated by identification of a helical collagen crossing site consisting of Hyl-Gly-His-Arg located at positions 87-90 in all collagen chains of this size thus far identified. Seventy-one percent homology (excluding Gly residues) was found between amino acids in this region of the alpha 1(XI) and of alpha 1(V) collagen chains while only 21 and 19% identity was calculated for the same region of alpha 2(V) and alpha 1(I) collagen chains, respectively.     

The thiol groups of mouse immunoglobulin A. Incomplete formation of the C alpha 1-domain disulphide bridge.

The BALB/c IgA (immunoglobulin A) myeloma protein M167 contained on average 5.7 free SH groups per IgA dimer. These groups were preponderantly on the heavy chains and comprised two distinct populations: 3.3 exposed SH groups per dimer in the Fc region, and 2.4 buried SH groups per dimer in the Fd region, detectable o only after denaturation. To locate the cysteine residues involved, labelled peptides were purified from thermolysin digests of radioalkylated IgA by high-performance liquid chromatography. From the amino acid compositions of the peptides, the exposed thiol groups were assigned to Cys-307 in the C alpha 2 domain, which thus existed in the reduced form to an extent exceeding 80%. This residue may allow attachment of secretory component to dimer IgA in the mouse to proceed via thiol-disulphide exchange. The buried thiol groups were assigned to Cys-150 and Cys-208, in the C alpha 1 domain, each being in the reduced form to the extent of approx. 30%. This pair of residues would normally give rise to the characteristic intradomain disulphide bridge. It appears that disulphide formation is not a crucial event during folding of the C alpha 1 domain in IgA biosynthesis. The sequence in the region 140-151 was re-investigated, and residue 142 was shown to be serine, not cysteine, helping explain the lack of heavy-chain-light chain bonding in BALB/c mouse IgA. A disulphide-bond model for mouse IgA is proposed on the basis of these assignments and other features of the mouse alpha-chain sequence.     

Primary structure of the elongation factor G from Escherichia coli. IX. Structure of peptides generated by cyanogen bromide cleavage of the G-factor isolated on thiol-activated sepharose and of the products of the G-factor cleavage at Asp-Pro bonds. Complete primary structure

The amino acid sequence of cysteine- and cystine-containing peptides resulting from cleavage of the G-factor by cyanogen bromide has been determined. For structure analysis cyanogen bromide peptides were further degradated using trypsin, chymotrypsin, thermolysin, staphylococcal glutamic protease, or limited acid hydrolysis. The products of the G-factor cleavage at Asp-Pro bonds were also studied. The obtained data together with those published earlier permitted to establish the complete primary structure of the elongation factor G. The polypeptide chain consists of 701 amino acid residues and has molecular mass of 77321,46.     

Amino acid sequence studies on the alpha chain of human fibrinogen. Covalent structure of the alpha-chain portion of fragment D.

The alpha-chain portion of fragment D has been purified from an exhaustive plasmic digest of human fibrinogen. The major polypeptide species has 91 amino acid residues, although a small amount of a 97-residue chain representing an earlier digestion stage remains. The amino acid sequence of the first 44 residues was determined by stepwise degradation with an automatic solid-phase sequencer. Another large stretch of sequence was revealed by the finding that the alpha chain of fragment D overlaps the cyanogen bromide fragments alphaCNIVA and alphaCNIII (Doolittle, R. F. Cassman, K. G., Cottrell, B. A., Friezner, S. J. Hucko, J. T., and Takagi, T. (1977), Biochemistry 16 (preceding paper in this issue)). The automatic sequencer results were confirmed and extended by the isolation and characterization of 18 of 19 expected tryptic peptides from the fragment D alpha chain. As a result, almost the entire sequence has been obtained. The overlap with key cyanogen bromide fragments has also allowed us to propose an order for the first 198 residues of the fibrinogen alpha chain. A striking homology with the gamma chain and beta chain is apparent which has interesting structural implications.     

Primary structure of streptococcal proteinase. II. Isolation, composition, and amino acid sequences of the tryptic and chymotryptic peptides of cyanogen bromide fragment 5.

The amino acid sequence of the cyanogen bromide fragment 5 of streptococcal proteinase has been determined. This fragment comprises residues 130 to 253 of the proteinase chain. Six tryptic peptides were isolated from maleylated cyanogen bromide fragment 5, and their alignment was obtained by the overlap of chymotryptic peptides. Sequence analysis of tryptic, chymotryptic, and thermolysin peptides was performed by the 5-deimethylaminoaphthalene-1-sulfonyl technique and carboxypeptidases digestion.     

Locus of a histidine-based, stable trifunctional, helix to helix collagen cross-link: stereospecific collagen structure of type I skin fibrils

The loci of the three amino acid residues that contribute their prosthetic groups to form the stable, nonreducible, trifunctional intermolecular cross-link histidinohydroxylysinonorleucine in skin collagen fibrils were identified. Two apparently homogeneous three-chained histidinohydroxylysinonorleucine cross-linked peptides were chromatographically isolated. They were obtained from a tryptic digest of denatured unreduced 6 M guanidine hydrochloride insoluble bovine skin collagen. Amino acid and sequence analyses demonstrated that the prosthetic groups of alpha 1(I)-chain Hyl-87, alpha 1(I)-chain Lys-16c, and alpha 2(I)-chain His-92 formed the cross-link. The latter results served to define the locus of the stable, nonreducible trifunctional moiety. Identical types of analyses were performed on the three-chained peptides isolated after bacterial collagenase digestion of the cross-linked tryptic peptides. This confirmed the initial identification and location of the three peptides linked by the cross-link. In addition, data reported here provide for a correction of the micromolecular structure for the alpha 2(I) chain. Stereochemical considerations concerning this trifunctional cross-link's specific locus indicate that the steric relationships between the alpha chains of skin and skeletal tissue collagens are fundamentally different and the intermolecular relationships in skin fibrils are specific for skin. The same molecular relationships also indicate that histidinohydroxylysinonorleucine links three molecules of collagen. The stereochemistry of cross-linking for skin collagen is in accordance with and explains the X-ray findings of a 65-nm periodicity found for this tissue [Stinson, R. H., & Sweeny, P. R. (1980) Biochim. Biophys. Acta 621, 158; Brodsky, B., Eikenberry, E. F., & Cassidy, K. (1980) Biochim. Biophys. Acta 621, 162].     

Rapid structural characterisation of a murine monoclonal IgA alpha chain: heterogeneity in the oligosaccharide structures at a specific site in samples produced in different bioreactor systems.

A murine hybridoma cell line which secretes a monoclonal IgA antibody, directed against the LPS antigen of Vibrio cholerae, was grown in either a continuous stirred tank reactor, a fluidised bed reactor or a hollow fibre reactor. Different methods were used for the structural characterisation of the IgA alpha chains. A classical approach consisted of Edman sequencing and mass determination of peptides separated by reversed phase HPLC. Alternatively, peptides and glycopeptides from a tryptic digest of each alpha chain were identified directly by MALDI-TOF mass spectrometry. A detailed analysis of the oligosaccharide structures at an unique site on the alpha chain was made by labelling the oligosaccharides released by N-glycosidase F with 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone. After separation by HPLC, mass measurements were made using matrix-assisted laser desorption time of flight mass spectrometry before and after digestion with specific exoglycosidases. The primary structure of the alpha chain of IgA was not affected by different cell culture conditions; in contrast, significant variations could be detected in the pattern of N-linked oligosaccharide structures, most prominently in the degree of sialylation. The efficiency of the analytical techniques in providing quality control of the identity, integrity and consistency of the glycoprotein is shown and discussed.     

Primary structure of the elongation factor G from Escherichia coli. V. Amino acid sequence of the C-terminal domain

The amino acid sequence of the C-terminal domain of the elongation factor G (EF-G) has been studied. The polypeptide chain of the domain consists of 228 amino acid residues, and contains no tryptophan or cysteine residues. To determine its structure, the peptides obtained as a result of the fragment digestion by staphylococcal glutamic protease, cyanogen bromide cleavage, and tryptic hydrolysis of the fragment modified by maleic anhydride have been analyzed, as well as peptides obtained after hydrolyses of cyanogen bromide fragments with chymotrypsin, thermolysin and trypsin.     

Primary structure of the L chain from a rabbit homogeneous antibody to streptococcal carbohydrate. I. Purification of antibody and sequence determination of peptides from alpha-chymo-tryptic and thermolytic digests.

Primary structural studies have been carried out on the light chain of a homogeneous rabbit antibody to Group C streptococcal carbohydrate. Approximately 20 g of this antibody were obtained by multiple exchange transfusions at the peak of the antibody response. The isolated antibody was homogeneous by several antigenic and chemical criteria. The light chain was isolated and modified, and then digested with alpha-chymotrypsin or thermolysin. The resulting peptides were isolated by gel filtration, paper electrophoresis, and paper chromatography. The amino acid sequences of these peptides were determined by Edman degradation plus dansylation. This supplied sufficient information to assign approximately 90 percent of the residues in the chain. The destruction of tyrosine during acid hydrolysis of peptides which had been eluted from a paper chromatogram was investigated. This destruction is due to inpurities in the paper which contaminate the peptides. Prevention of such destruction can be achieved by predevelopment of the paper with 1 N NH4OH prior to paper chromatography.     

Determination of the disulfide bond pairings in human tissue factor pathway inhibitor purified fromEscherichia coli

The disulfide bond assignments of human alanyl tissue factor pathway inhibitor purified fromEscherichia coli have been determined. This inhibitor of the extrinsic blood coagulation pathway possesses three Kunitz-type inhibitor domains, each containing three disulfide bonds. The disulfide bond pairings in domains 1 and 3 were determined by amino acid sequencing and mass spectrometry of peptides derived from a thermolysin digest. However, thermolysin digestion did not cleave any peptide bonds within domain 2. The disulfide bond pairings in domain 2 were determined by isolating it from the thermolysin treatment and subsequently cleaving it with pepsin and trypsin into peptides which yielded the three disulfide bond pairings in this domain. These results demonstrate that the disulfide pairings in each of the three domains of human tissue factor pathway inhibitor purified fromEscherichia coli are homologous to each other and also to those in bovine pancreatic trypsin inhibitor.     

Surface localization of apolipoprotein AII in lipoprotein-complexes.

Lipoprotein particles reconstituted from the apolipoprotein AII (apo AII) component of human serum high density lipoprotein, phosphatidylcholine and lysophosphatidylcholine were covalently linked to the imidoester groups of a polystyrene residue. Apo AII was proteolytically digested with thermolysin after delipidation. The covalently bound peptides remaining at the resin were cleaved and separated by combined two-dimensional electrophoresis/chromatography. The peptides were isolated, hydrolyzed and their amino acid composition determined. They were assigned to the apo AII sequence. Since the imidoester groups on the surface of the resin carrier cannot react with buried lysine residues, this method gives strong chemical evidence for the spreading of the apo AII polypeptide chain over the surface of the lipoprotein particle, as far as the sequence carrying lysine residues between residue 22 and 55 of each symmetrical half is concerned.     

Determination of the disulfide bond pairings in human tissue factor pathway inhibitor purified fromEscherichia coli

The disulfide bond assignments of human alanyl tissue factor pathway inhibitor purified fromEscherichia coli have been determined. This inhibitor of the extrinsic blood coagulation pathway possesses three Kunitz-type inhibitor domains, each containing three disulfide bonds. The disulfide bond pairings in domains 1 and 3 were determined by amino acid sequencing and mass spectrometry of peptides derived from a thermolysin digest. However, thermolysin digestion did not cleave any peptide bonds within domain 2. The disulfide bond pairings in domain 2 were determined by isolating it from the thermolysin treatment and subsequently cleaving it with pepsin and trypsin into peptides which yielded the three disulfide bond pairings in this domain. These results demonstrate that the disulfide pairings in each of the three domains of human tissue factor pathway inhibitor purified fromEscherichia coli are homologous to each other and also to those in bovine pancreatic trypsin inhibitor.     

The primary structure of staphylococcal protease.

The amino acid sequence of staphylococcal protease has been determined by analysis of tryptic peptides obtained from cyanogen bromide fragments. Selected peptides obtained from digests with staphylococcal protease, thermolysin, and chymotrypsin provided the information necessary to align the tryptic peptides and the cyanogen bromide fragments. The protease is a single polypeptide chain of some 250 amino acids and is devoid of sulfhydryl groups. The COOH-terminal tryptic peptide of of the protease molecule contains some 43 residues, most of which are aspartic acids, asparagines, and prolines. The amino acid sequence of this peptide was not determined. The primary structure near the active serine residue indicates that staphylococcal protease is related to the pancreatic serine proteases. However, it has little or no additional sequence homologies with these enzymes except for the regions near histidine-50 and aspartic acid - 91. These regions have striking similarities with the corresponding regions of protease B and the trypsin-like enzyme of Streptomyces griseus.     

The primary structure of the calcium ion-transporting adenosine triphosphatase protein of rabbit skeletal sarcoplasmic reticulum. Peptides derived from digestion with cyanogen bromide, and the sequences of three long extramembranous segments.

The isolation and characterization of the soluble peptides from the CNBr digest of the calcium ion-transporting adenosine triphosphatase protein of rabbit skeletal sarcoplasmic reticulum are described. The 562 unique residues of the protein were placed in sequences. The remaining part of the protein (about 500 residues) yielded long hydrophobic sequences that contained all but one of the tryptophan residues of the protein and that were probably derived largely from the intramembranous parts of the protein. Three long stretches of primary structure, constituting half of the protein, have been reconstructed from the information presented here together with the sequences found in peptides from other digests of the protein. The secondary structures of these sequences have been predicted. A model for the primary structure of the protein is presented and the implications discussed. Details of the isolation of peptides are contained in Supplementary Publication, SUP 50105 (29 pages), which has been deposited with 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. (1978) 169, 5.     

Tubulin structure probed with antibodies to synthetic peptides. Mapping of three major types of limited proteolysis fragments

We synthesized five peptides homologous to the potentially antigenic positions alpha(214-226), alpha(430-443), alpha(415-443), beta(241-256), and beta(412-431) of the porcine brain tubulin sequences. These peptides were successfully employed to raise tubulin-cross-reactive antibodies. The antibodies are specific of the regions of tubulin spanned by the peptides. They react specifically with the tubulin bands in immunoblots and with microtubules in immunofluorescence assays of cytoskeletons. The peptides of the C-terminal regions have also been employed to localize determinants recognized by two available monoclonal antibodies to tubulin in the positions alpha(415-430) and beta(412-431), respectively. In a first application of the anti-peptide antibodies, we have mapped the fragments of limited proteolysis of purified calf brain tubulin by trypsin, chymotrypsin, papain, thermolysin, subtilisin, and protease V8 from Staphylococcus aureus. Thirty-seven peptides have been identified, of which 32 have been unequivocally aligned into the tubulin sequences on the basis of their antigenic reactivity. There are three major, well-defined zones of preferential cleavage by the proteases: the C-termini and two internal zones in each chain. C-Terminal cleavages of both chains by subtilisin do not remove the antigenic reactivity of the zones alpha(415-430) and beta(412-431). C-Terminal cleavages by protease V8 are preferential of beta-tubulin. All six proteases tested cleave alpha- and/or beta-tubulin at one or both of the internal zones. These zones are located roughly at one-third and two-thirds of the chain length in both subunits. Therefore, a model of the tubulin monomers is proposed which consists of three major, proteolytically defined, compact regions (N-terminal, middle, and C-terminal thirds) and the cleavable zones. This model is discussed with the tubulin structural information presently available.     

Histidine decarboxylase of Lactobacillus 30a. Sequences of the overlapping peptides, the complete alpha chain, and prohistidine decarboxylase

The complete amino acid sequence of the alpha chain of histidine decarboxylase of Lactobacillus 30a has been established by isolation and analysis of the eight methionine-containing tryptic peptides of this chain. These peptides provide the overlaps required to order all nine peptides derived by complete cyanogen bromide cleavage of the alpha chain (Huynh, Q.K., Vaaler, G.L., Recsei, P.A., and Snell, E.E. (1984) J. Biol. Chem. 259, 2826-2832). Ordering of six of the latter peptides was confirmed by isolation and analysis of four peptides derived by incomplete cyanogen bromide cleavage. The alpha chain is composed of 226 residues and has a molecular weight of 24,892 calculated from the sequence. These results and the previously determined sequence of the beta chain (Vaaler, G.L., Recsei, P.A., Fox, J.L., and Snell, E.E. (1982) J. Biol. Chem. 257, 12770-12774) establish the complete amino acid sequence of the enzyme and of the pi chain of prohistidine decarboxylase. The latter is composed of 307 amino acids and has a calculated molecular weight of 33,731. Four segments of the pi chain sequence are repeated. The bond between Ser-81 and Ser-82 that is cleaved during proenzyme activation is in an uncharged portion of the sequence that is rich in serine and threonine residues and is predicted to be part of a beta sheet structure.