Sulfation of a gamma-chain variant of human fibrinogen

Biosynthetic sulfation of human fibrinogen was investigated using a hepatoma-derived cell line in culture. Very little [35]sulfate was incorporated into the major forms of the A alpha, B beta, or gamma-chains of fibrinogen, but there was a labeled peptide chain with electrophoretic mobility intermediate between the B beta and gamma-chains. Base hydrolysis of the sulfate-labeled product released tyrosine sulfate. The labeled peptide is identified as a form of gamma-chain by its resistance to proteolysis during extended periods of incubation, in contrast with A alpha and B beta-chains which are converted to smaller forms. The results indicate that human fibrinogen contains tyrosine sulfate primarily within a variant form of the gamma-chain.     

Identification of the site of sulfation of the fourth component of human complement

The alpha-chain of the fourth component of complement (C4) contains tyrosine sulfate (Karp, D.R. (1983) J. Biol. Chem. 258, 12745-12748). Here we have determined the site and stoichiometry of sulfation of C4 secreted by the human hepatoma-derived cell line Hep G2. C4 was labeled with [35S]sulfate and isolated from culture medium by immunoprecipitation. C4 digested with trypsin and chymotrypsin and analyzed by reverse-phase high-performance liquid chromatography contained a single sulfate-labeled peptide. Digestion of C4 with trypsin alone yielded two major sulfate-labeled peptides, suggesting that there may be some sequence variability in C4 near the site of sulfation. Sequential Edman degradation of tryptic peptides labeled with [3H]tyrosine and [35S]sulfate detected tyrosine residues at positions 5, 13, 16, and 18. Chymotrypsin cleaved 5 residues off the NH2-terminal end of tryptic peptides, yielding a peptide with tyrosine at positions 8, 11, and 13. Comparison of the position of tyrosine residues with the reported sequence of C4 identified the sites of sulfation as tyrosine residues at positions 738, 741, and 743 in the alpha-chain of C4. All 3 of these tyrosine residues appeared to be sulfated. When sulfation of C4 was partially inhibited by addition of catechol to culture medium, three different forms of the peptide were resolved by high-performance liquid chromatography, consistent with peptides containing 1, 2, or 3 sulfates. Comparison of the quantities of tyrosine and tyrosine sulfate in C4 which had been labeled with [3H]tyrosine and digested with Pronase also indicated that C4 contained an average of 2-3 residues of tyrosine sulfate/molecule. These results suggest that the biologically active form of the protein is sulfated.     

Identification of two sites of sulfation of human heparin cofactor II

Tyrosine sulfate was identified as a constituent of human heparin cofactor II by analysis of sulfate-labeled protein secreted by a human hepatoma-derived cell line and of purified protein from human plasma. Alkaline hydrolysis of heparin cofactor II released tyrosine sulfate as demonstrated by anion-exchange high performance liquid chromatography of hydrolysates. Two sites of sulfation were identified, and the amino acid sequences of the sites were established by sequential Edman degradation of sulfate-containing tryptic peptides that were isolated by reverse-phase high performance liquid chromatography. Each peptide contains only a single tyrosine residue so that the sites of sulfation can be assigned unambiguously. The two sites of sulfation are separated by 13 residues and represent an internal sequence repeat in the heparin cofactor II molecule. The two sites have the following sequences. Glu56-Asp-Asp-Asp-Tyr(SO4)-Leu-Asp62 Glu69-Asp-Asp-Asp-Tyr(SO4)-Ile-Asp75 Sulfate-labeled heparin cofactor II formed a covalent complex with thrombin in a heparin-dependent manner. Thus, the sulfate-containing form of the protein was shown to be biologically active. The characteristic sulfate-containing segment of heparin cofactor II, which contains 17 acidic amino acid residues over a span of 30 residues, may contribute to the unique properties of this thrombin inhibitor.     

Primary structure of human alpha 2-antiplasmin, a serine protease inhibitor (serpin)

The plasma protein alpha 2-antiplasmin is the main physiological inhibitor of the serine protease plasmin, which is responsible for the dissolution of fibrin clots. We have determined the primary structure of mature human alpha 2-antiplasmin by DNA sequencing of overlapping cDNA fragments prepared from human liver mRNA. cDNA clones were identified by hybridization with a 48-base pair deoxyoligonucleotide probe deduced from the sequence of a 16-amino acid peptide of alpha 2-antiplasmin. Mature human alpha 2-antiplasmin contains 452 amino acids. It is homologous (23-28%) with five other proteins belonging to the serine protease inhibitor (serpin) superfamily. Its reactive site, i.e. the peptide bond cleaved by reaction with its primary target enzyme, plasmin, consists of Arg364-Met365. This dipeptide corresponds to the reactive site Met358-Ser359 of the archetypal serpin, alpha 1-antitrypsin.     

Identification and functional importance of tyrosine sulfate residues within recombinant factor VIII.

Sulfated tyrosine residues within recombinant human factor VIII were identified by [35S]sulfate biosynthetic labeling of Chinese hamster ovary cells which express human recombinant factor VIII. Alkaline hydrolysis of purified [35S]sulfate-labeled factor VIII showed that greater than 95% of the [35S]sulfate was incorporated into tyrosine. [3H]Tyrosine and [35S]sulfate double labeling was used to quantify the presence of 6 mol of tyrosine sulfate per mole of factor VIII. Amino acid sequence analysis of thrombin and tryptic peptides isolated from [35S]sulfate-labeled factor VIII demonstrated tyrosine sulfate at residue 346 in the factor VIII heavy chain and at residues 1664 and 1680 in the factor VIII light chain. In addition, the carboxyl-terminal half of the A2 domain contained three tyrosine sulfate residues, likely at positions 718, 719, and 723. Interestingly, all sites of tyrosine sulfation border thrombin cleavage sites. The functional importance of tyrosine sulfation was examined by treatment of cells expressing factor VIII with sodium chlorate, a potent inhibitor of tyrosine sulfation. Increasing concentrations of sodium chlorate inhibited sulfate incorporation into factor VIII without affecting its synthesis and/or secretion. However, factor VIII secreted in the presence of sodium chlorate exhibited a 5-fold reduction in procoagulant activity, although the protein was susceptible to thrombin cleavage. These results suggest that tyrosine sulfation is required for full factor VIII activity and may affect the interaction of factor VIII with other components of the coagulation cascade.     

Amino acid sequence studies on the alpha chain of human fibrinogen. Overlapping sequences providing the complete sequence

The complete amino acid sequence of the alpha chain of human fibrinogen has been determined. It contains 610 amino acid residues and has a calculated molecular weight of 66,124. The chain has 10 methionines, and fragmentation with cyanogen bromide yields 11 peptides [Doolittle, R.F., Cassman, K.G., Cottrell, B.A., Friezner, S.J., Hucko, J.T., & Takagi, T. (1977) Biochemistry 16, 1703]. The arrangement of the 11 fragments was determined by the isolation of peptide overlaps from plasmic and staphylococcal protease digests of fibrinogen and/or alpha chains. In addition, certain of the cyanogen bromide fragments, preliminary reports of whose sequences have appeared previously, have been reexamined in order to resolve several discrepancies. The alpha chain is homologous with the beta and gamma chains of fibrinogen, although a large repetitive segment of unusual composition is absent from the latter two chains. The existence of this unusual segment divides the sequence of the alpha chain into three zones of about 200 residues each that are readily distinguishable on the basis of amino acid composition alone.     

Cyanogen bromide digestion of the avian myeloblastosis virus pp19 protein: isolation of an amino-terminal peptide that binds to viral RNA.

The avian myeloblastosis virus pp19 protein was separated from the other virus proteins by a rapid and simple purification procedure which yields milligram amounts of homogeneous protein. This protein was then fragmented by digestion with cyanogen bromide. When the mixture of the cyanogen bromide peptides was passed through a 60S avian myeloblastosis virus RNA-cellulose column, only one peptide bound with high affinity to the resin. The peptide migrated on a sodium dodecyl sulfate-polyacrylamide gel with an approximate molecular weight of 2,900 and will be referred to as the p3B peptide. This peptide was also isolated directly by chromatography of the cyanogen bromide-digested pp19 protein on a reverse-phase high-pressure liquid chromatography column. It was again the only cyanogen bromide peptide of the pp19 protein that bound to the RNA affinity resin. The p3B peptide is a basic peptide, as was seen by its rapid migration on acid-urea-polyacrylamide gels and its amino acid composition. A partial amino acid sequence analysis of the p3B peptide indicated that it was derived from the amino terminus of the intact protein. Although the p3B peptide bound to 60S RNA, it did not demonstrate the selective binding of native pp19 to regions of the RNA containing secondary structure.     

Amino acid sequence studies on the alpha chain of human fibrinogen. Location of four plasmin attack points and a covalent cross-linking site.

The amino acid sequence of a 38-residue midsection piece of the alpha chain of human fibrinogen has been determined using a combination of plasmin-derived peptides and cyanogen bromide fragments. The segment contains several important features, including four early plasmin attack points, one of the two alpha-chain cross-linking acceptor sites, and a peptide homologous to one isolated from plasmin digests of bovine fibrinogen, and reported to have anticoagulant activity. The segment is sequentially adjacent to and overlapping with a large molecular weight (20000-25000) fragment released during plasminolysis. This latter material is very rich in glycine and serine and deficient in nonpolar amino acids. It also contains the other alpha-chain cross-linking acceptor site.     

The tryptophan synthase alpha 2 beta 2 complex: a comparison of the reactivity of amino groups in the alpha and beta 2 subunits and in the complex by differential labeling studies

The interaction of the alpha and beta 2 subunits of tryptophan synthase of Escherichia coli to form an alpha 2 beta 2 complex has been probed by differential labeling studies. In the first step the separate alpha or beta 2 subunit or the alpha 2 beta 2 complex was labeled by reductive methylation with trace amounts of [3H]HCHO in the presence of NaCNBH3. In the second step the 3H-labeled preparation was fully labeled under denaturing conditions with [14C]HCHO and NaCNBH3. Peptides containing labeled monomethyl or dimethyl amino groups were isolated after thermolytic digestion or after cyanogen bromide treatment. The 3H/14C ratio of each peptide is a measure of the relative reactivity of the amino group or groups in each peptide. The most reactive amino group in the alpha subunit, lysine-109, is strongly shielded from modification in the alpha 2 beta 2 complex. The most reactive amino group in the beta 2 subunit, the amino-terminal threonine, is not shielded from modification in the alpha 2 beta 2 complex.     

The isolation and characterization of the cyanogen bromide peptides from the B chain of human collagen.

Cleavage of the collagen B chain with cyanogen bromide yields nine peptides which have been isolated and characterized with regard to molecular weight and amino acid composition. The peptides are recovered in equimolar quantities and account for the full amino acid complement of the chain as isolated following limited pepsin digestion of human placental tissue. These data thus confirm the unique composition of the chain and further indicate that the chain has been isolated in essentially pure form. The total number of amino acid residues (1018) observed in the cyanogen bromide peptides of the B chain indicate that it is comparable in length to the previously characterized collagen alpha chains. Thus, the apparent larger size of the B chain noted in previous studies may possibly be attributed to the relatively large quantities of hydroxylysine-linked carbohydrate, but more likely to the increased numbers of large hydrophobic amino acids in the B chain. Although the cyanogen bromide peptide pattern obtained in studies on the B chain serves to differentiate this chain from other known chains, some possible homologies between the B chain peptides and peptides derived from the alpha chains of type I, II, and III collagens are noted.     

Outer membrane proteins of Escherichia coli. V. Evidence that protein 1 and bacteriophage-directed protein 2 are different polypeptides.

Protein 1 from the outer membrane of Escherichia coli K-12 and protein 2 from a phage PA-2 lysogen of the same strain were isolated by differential sodium dodecyl sulfate extraction and purified by ion-exchange and gel filtration chromatography. Rabbit antisera were prepared against these proteins and showed no cross-reaction between proteins 1 and 2. The proteins have the same N-terminal amino acid but show small yet significant differences in amino acid composition. The proteins were cleaved with cyanogenbromide in solvents containing both formic acid and trifluoroacetic acid. By comparing the cleavage in these solvents, it was established that protein 1 yielded 5 cyanogen bromide peptides, and the sum of the molecular weights of these was equivalent to the molecular weight of the uncleaved protein. Protein 2 yielded 4 cyanogen bromide peptides, none of which was identical to those of protein 1, and the sum of these peptides was also equivalent to the apparent molecular weight of the uncleaved protein. Significant differences were also observed when tryptic peptides from the two proteins were compared. These results indicate that protein 1 and the phage-directed protein 2 are distinct, different, and apparently homogeneous proteins.     

Human plasma prekallikrein, a zymogen to a serine protease that contains four tandem repeats

The amino acid sequence of human plasma prekallikrein was determined by a combination of automated Edman degradation and cDNA sequencing techniques. Human plasma prekallikrein was fragmented with cyanogen bromide, and 13 homogeneous peptides were isolated and sequenced. Cyanogen bromide peptides containing carbohydrate were further digested with trypsin, and the peptides containing carbohydrate were isolated and sequenced. Five asparagine-linked carbohydrate attachment sites were identified. The sequence determined by Edman degradation was aligned with the amino acid sequence predicted from cDNAs isolated from a lambda gt11 expression library. This library contained cDNA inserts prepared from human liver poly(A) RNA. Analysis of the cDNA indicated that human plasma prekallikrein is synthesized as a precursor with a signal peptide of 19 amino acids. The mature form of the protein that circulates in blood is a single-chain polypeptide of 619 amino acids. Plasma prekallikrein is converted to plasma kallikrein by factor XIIa by the cleavage of an internal Arg-Ile bond. Plasma kallikrein is composed of a heavy chain (371 amino acids) and a light chain (248 amino acids), and these 2 chains are held together by a disulfide bond. The heavy chain of plasma kallikrein originates from the amino-terminal end of the zymogen and is composed of 4 tandem repeats that are 90 or 91 amino acid residues in length. These repeat sequences are also homologous to those in human factor XI. The light chain of plasma kallikrein contains the catalytic portion of the enzyme and is homologous to the trypsin family of serine proteases.     

Peptide mapping by CNBr fragmentation using a sodium dodecyl sulfate-polyacrylamide minigel system

The method of peptide mapping by sodium dodecyl sulfate-gel electrophoresis following partial protein fragmentation with cyanogen bromide was adapted for a polyacrylamide minigel system. The combined use of the discontinuous gel electrophoresis system of J. P. Doucet and J. M. Trifaró [1988) Anal. Biochem. 168, 265-271) and a vertical polyacrylamide minigel system produced the following advantages over other procedures: (a) the ability to resolve cyanogen bromide cleavage fragments over a broad molecular mass range while yielding very sharp protein staining bands; (b) well-defined peptide maps are produced with as little as 2 micrograms of protein; (c) less time is required to perform fragmentation with cyanogen bromide, to equilibrate the gel slices in sodium dodecyl sulfate buffer, as well as to perform the electrophoresis; and (d) the cyanogen bromide fragmentation patterns are highly reproducible.     

Partial amino acid sequence of human plasma retinol-binding protein. Isolation and alignment of the five cyanogen bromide fragments and the amino acid sequences of four of the fragments

Studies are reported on the primary structure of human retinol-binding protein (RBP), the specific plasma transport protein for vitamin A. The protein consists of a single polypeptide chain of 186-187 amino acids. RBP was cleaved by cyanogen bromide into five fragments, CB-I (27 residues), CB-11 (25 residues), CB-III (20 residues), CB-IV (15 residues), and CB-V (99-100 residues). The cyanogen bromide fragments were isolated, their compositions were determined, and they were aligned after studies that included the tryptic digestion of maleylated, reduced, and carboxymethylated RBP and subsequent enzymatic digestion of some of the resulting tryptic peptides. The amino acid sequences of four of the five cyanogen bromide fragments were determined, and the sequence of almost two-thirds of the NH2-terminal portion of the RBP molecule was determined as: H2N-GLU-Arg-Asp-Cys-Arg-Val-Ser-ser-Phe-Arg-Val-Lys-Glu-Asn-Phe-Asp-Lys-Ala-Arg-Phe-Ser-Gly-Thr-Trp-Tyr-Ala-Met-Ala-Lys-Lys-Asp-Pro-Glu-Gly-Leu-Phe-Leu-Gln-Asp-Asx-Ile-Val-Ala-Glu-Phe-Ser-Val-Asx-Glx-Gly-Thr-Met-Ser-Ala-Thr-Ala-Gly-Lys-Arg-Val-Arg-Leu-Leu-Asn-Asn-Trp-Asp-Val-Cys-Ala-Asp-Met-Val-Gly-thr-Phe-Thr-Asp-Thr-Glu-Asp-Pro-Ala-Lys-Phe-Lys-Met-Lys-Tyr-Trp-Gly-Val-Ala-Ser-Phe-Leu-Gln-Lys-Gyl-Asn-Asp-Asx-His-Trp-Ile-Val-Asp-Thr-Asx-Thr-Tyr-Tyr-Ala-Val-Glu-Tyr-Cys-Ser-Arg---.     

Phosphofructokinase: complete amino-acid sequence of the enzyme from Bacillus stearothermophilus

The entire amino acid sequence of the protein subunit of phosphofructokinase from Bacillus stearothermophilus has been established mainly by sequence analysis of cyanogen bromide fragments and of peptides derived from these fragments by further digestion with proteolytic enzymes. Overlaps of the cyanogen bromide fragments as well as peptide sequences necessary to complement and to confirm tentative assignments within the larger peptide fragments were obtained from the sequences of selected peptides isolated from tryptic and chymotryptic digests of the intact S-[14C]-carboxymethylated protein. Sequence information was also provided by automated sequence analysis of the intact protein subunit and of some of the larger peptide fragments. The sequence is as follows: (See Text).     

The amino acid sequence of Clostridium pasteurianum iron protein, a component of nitrogenase. II. Cyanogen bromide peptides

A total of 10 cyanogen bromide peptides were isolated from the S-beta-carboxymethyl iron protein of nitrogenase. Purification of these peptides was performed mainly by gel filtration on Sephadex G-50; by ascending paper chromatography using the solvent system of pyridine, isoamyl alcohol, 0.1 M ammonium hydroxide; and also, in some cases, with additional steps such as anion exchange column chromatography on Dowex 1-X2 or ascending paper chromatography in an acidic solvent system or by pyridine precipitation of the cyanogen bromide fragment. Sequenator analyses of three large cyanogen bromide peptides (53 to 72 residues) provided tryptic peptide overlap data for the inner portion of the protein. The cyanogen bromide peptides accounted for all of the 273 amino acid residues which were present in the tryptic peptides isolated from carboxymethyl-iron protein (Tanaka, M., Haniu, M., Yasunobu, K. T., and Mortenson, L. E. (1977) J. Biol. Chem. 252, 7081-7088).     

Covalent modification of an adenosine 3':5'-monophosphate binding site of the regulatory subunit of cAMP-dependent protein kinase II with 8-azidoadenosine 3':5'-monophosphate. Identification of a single modified tyrosine residue

The regulatory subunit of cAMP-dependent protein kinase II (RII) from porcine heart was modified specifically and covalently using the photoaffinity reagent, 8-azidoadenosine 3':5'-monophosphate (8-N3cAMP). In the presence of excess cAMP, the photo-dependent incorporation of 8-N3cAMP was abolished whereas excess AMP and ATP had no effect. A maximum incorporation of 0.5 mol of 8-N3cAMP was achieved/mol of regulatory subunit monomer (Mr = 55,000). This level of incorporation was obtained when the purified regulatory subunit was treated with urea prior to labeling to remove residual bound cAMP. When the regulatory subunit was labeled with radioactive 8-N3cAMP, cleaved with trypsin, and the tryptic peptides mapped in two dimensions, a single major radioactive peptide was observed. Chemical cleavage of the radioactively labeled RII with cyanogen bromide and subsequent chromatography on Sephadex G-50 also yielded a single major peak of radioactivity. The covalently modified cyanogen bromide peptide subsequently was purified to homogeneity using high performance liquid chromatography. Greater than 90% of the radioactivity that was incorporated into the regulatory subunit was recovered in this cyanogen bromide peptide which had the following sequence: Lys-Arg-Asn-Ile-Ser-His-Tyr (cAMP)-Glu-Glu-Cln-Leu-Val-Lys-Hse. When the Edman degradation of this peptide was carried out, the radioactivity derived from the 8-N3cAMP was released with the tyrosine residue at Step 7 identifying this residue as the specific site of attachment of the photoaffinity reagent.     

Primary structure of tyrosinase from Neurospora crassa. II. Complete amino acid sequence and chemical structure of a tripeptide containing an unusual thioether

To align the four cyanogen bromide peptides of Neurospora tyrosinase whose amino acid sequences were reported in the preceding paper, suitable methionine-containing overlap peptides were isolated. The required peptides were obtained by tryptic, peptic, and thermolytic digestion of the unmodified protein and of the maleylated derivative. From the partial sequence information of these peptides and a cyanogen bromide overlap peptide, the four cyanogen bromide fragments were aligned in the order CB3-CB1-CB4-CB2. These data establish Neurospora tyrosinase as a single-chain protein of 407 amino acids with a molecular weight of 46,000. The single cysteinyl residue 94 was found to be covalently linked via a thioether bridge to histidyl residue 96. The chemical nature of this unusual structure was elucidated by physicochemical analysis of peptides obtained from in vivo 35S, [2,5-3H]histidine, and [5-3H]histidine-labeled Neurospora tyrosinase.     

Structural studies of lens fiber junction protein MP26 by cyanogen bromide cleavage

The lens fiber-cell plasma membrane MP26 from chick, bovine, and human lenses yielded identical cyanogen bromide peptide maps, confirming the essential conservation of structure in the junction protein of vertebrate lens fiber cells. Immunoblot analyses of the cyanogen bromide peptide maps of human lens MP26 and of its age-dependent proteolytic product MP22 confirmed that MP22 is a derivative of MP26. The findings in this study are the first consistent with the positioning of the methionine residues in lens MP26 as predicted by its cDNA-derived sequence.     

The complete amino acid sequence of proapolipoprotein A-I of chicken high density lipoproteins

The complete amino acid sequence of proapolipoprotein (proapo) A-I of chicken high density lipoproteins was determined by sequencing overlapping peptides produced by trypsin, S. aureus V8 protease, and cyanogen bromide cleavage. There are 240 amino acid residues in mature chicken apoA-I. By direct sequence analysis of a cyanogen bromide peptide, we also determined the sequence of a 6-amino-acid prosegment which is present at approx. 10% the molar amount of the mature peptide in chicken plasma. Sequence comparison among apoA-I from chicken, human, rabbit, dog and rat, and secondary structure analysis indicate that while the degree of sequence homology is only moderate (less than 50% between chicken and man), there is good conservation of apoA-I secondary structure, especially in the N-terminal two-thirds of the protein in these widely separated species.