Complement component C6 is known to contain two factor I modules in tandem at its C-terminus. To localize the disulfide bridges in those domains, native C6 was cleaved with trypsin, followed by subtilisin. The resulting digests were separated by reversed-phase HPLC, and all of the potential cystine-containing fragments were detected by a fluorescence assay and amino acid composition analyses. Final identification of the disulfide bonds was achieved by Edman degradation of the corresponding peptides. From the data gained a 1–3, 2–9, 4–7, 5–10, 6–8 pattern was determined (Cys752–Cys802, Cys763–Cys780, Cys765–Cys816, Cys772–Cys795, Cys841–Cys852, Cys846–Cys898, Cys859–Cys876, Cys861–Cys911, Cys867–Cys891). These findings are compared with the strongly related complement components C7 and factor I.

The plasminogen kringle 2 (K2HPg) and kringle 3 (K3HPg) modules occur in tandem within the polypeptide segment that affords the heavy chain of plasmin. The K2HPg and K3HPg are unique among the plasminogen kringle domains in that they also are linked to each other via the Cys169-Cys297 (Cys4 of K2HPg to Cys43 of K3HPg, kringle numbering convention) disulfide bridge, thus generating a K2HPg-K3HPg "supermodule". The kringle (2 + 3) sequence of human plasminogen (r-EE[K2HPgK3HPg]DS) was expressed in Escherichia coli, using an expression vector containing the phage T5 promoter/operator N250PSN250P29 and the codons for an N-terminal hexahistidine tag to ensure the isolation of the recombinant protein by affinity chromatography on Ni(2+)-nitrilotriacetic acid/agarose under denaturing and reducing conditions. Kringle (2 + 3) was refolded in the presence of glutathione redox buffer. By taking advantage of the lysine affinity of kringle 2, the protein was purified by affinity chromatography on lysine-Bio-Gel. Recombinant kringle (2 + 3) was identified by amino acid composition, N-terminal sequence and mass determination. The 1H NMR spectrum shows that the intact r-K2HPgK3HPg is properly folded. By reference to spectra of the individual kringles, r-K2HPg and r-K3HPg, resonances of the K2HPg and K3HPg components in the spectrum of the intact r-K2HPgK3HPg can be readily distinguished. The strictly conserved Leu46 residue (kringle residue number convention) yields delta-methyl signals that are characteristic for K2HPg and K3HPg, exhibiting chemical shifts of -0.87 and -0.94 ppm, respectively, which are distinct from those of K1HPg, K4HPg, and K5HPg, (-1.04 to -1.05 ppm). Thus, the high-field Leu46 signals from K2HPg and K3HPg are well resolved from those of other kringles and can be identified unambiguously in spectra of the K1HPgK2HPgK3HPg elastolytic fragment of plasminogen as well as in spectra of Glu-plasminogen. Overall, r-K2HPgK3HPg exhibits broader resonance line widths than does the K1HPg component, consistent with a lesser mobility of the K2HPgK3HPg segment within the K1HPgK2HPgK3HPg fragment, a reflection of the extra structural constraint imposed by the disulfide bridge linking K2HPg to K3HPg. The ligand 6-aminohexanoic acid (6-AHA), which is known to interact with r-K2HPg but not with r-K3HPg, selectively perturbs K2 aromatic signals in the intact r-K2HPgK3HPg spectrum while leaving K3 resonances largely unaffected. Association constant (K(a)) values for 6-AHA determined from 1H NMR ligand titration experiments yield K(a) approximately 2.2 +/- 0.3 mM(-1) for the intact r-K2HPgK3HPg, comparable to K(a) approximately 2.3 +/- 0.2 mM(-1) determined for the isolated r-K2HPg, which demonstrates that the interactions of 6-AHA with the K2HPg ligand-binding site are not significantly affected by the neighboring K3HPg domain within the intact r-K2HPgK3HPg supermodule.

C9 is the most abundant protein of the membrane attack complex of complement. By means of limited proteolysis, different chromatographic techniques, a thiol-specific fluorescence assay, amino acid analysis, and Edman degradation 9 out of 12 disulfide bridges are definitely assigned (Cys22-Cys57, Cys33-Cys36, Cys67-Cys73, Cys121-Cys160, Cys233- Cys234, Cys359-Cys384, Cys489-Cys505, Cys492-Cys507, Cys509-Cys518). Weaker evidence permits to reduce the number of possible configurations for the remaining 3 cystines (Cys80-Cys91, Cys86-Cys104, Cys98-Cys113, or Cys80-Cys91, Cys86-Cys113, Cys98-Cys104). These findings are discussed in comparison with the strongly related components C6, C7, C8alpha, and C8beta.

The kringle 2 (E161T/C162S/EEE[K2HPg/C169S]TT) and the kringle 3 (TYQ[K3HPg]DS) domains of human plasminogen (HPg) were expressed in Escherichia coli in an expression vector with the phage T5 promotor/operator element N250PSN250P29 and the cDNA sequence for a hexahistidine tail to facilitate the isolation of the recombinant protein. A coagulation factor Xa (FXa)-sensitive cleavage site was introduced to remove the N-terminal histidine tag. In r-K2, mutations E161T and C162S were introduced to enhance the FXa cleavage yield and C169S to replace the cysteine residue, participating in the inter-kringle disulfide bridge between kringles 2 and 3. Recombinant proteins were isolated by affinity chromatography on Ni(2+)-nitrilotriacetic acid/agarose and refolded under denaturing and reducing conditions followed by a non-denaturing and oxidising environment. The free thiol group in position 297 in r-K3 was selectively alkylated with iodoacetamide. The hexahistidine tail was successfully removed with FXa. The N-terminal sequence, the amino acid composition and the molecular mass analyses are in agreement with the expected data. The correct arrangement of the disulfide bonds was verified by sequence analysis of the corresponding thermolytic and subtilisin fragments. r-K2 exhibits weak binding to lysine-Bio-Gel. The weak binding affinity of r-K2 for omega-aminocarboxylic acids is confirmed by intrinsic fluorescence titration with 6-aminohexanoic acid (NH2C5COOH) indicating a Kd of approximately 401 microM. In contrast, r-K3 seems to be devoid of a binding affinity for omega-aminocarboxylic acids. Considering earlier determined Kd values of kringle 1, kringle 4 and kringle 5, the binding affinity of HPg kringle domains for NH2C5COOH is proposed to decrease in the following order, kringle 1 > kringle 4 > kringle 5 > kringle 2 > kringle 3.

C1s, one of the three subcomponents of C1, the first component of the complement system, is a complex serine protease. To determine the disulfide-bonding pattern, fragments of C1s were generated by cleavage with pepsin, thermolysin, or subtilisin. Disulfide bonds have been identified by several methods, for example, direct observation of the phenylthiohydantoin derivative of cystine during Edman degradation of isolated peptides and placement in the known cDNA sequence. All of the 26 half-cystines are linked in disulfide bonds occurring at positions 50-68, 120-132, 128-141, 143-156, 160-187, 219-236, 279-326, 306-339, 344-388, 371-406, 410-534, 580-603, and 613-644. All of the disulfide bonds of the earlier described substructures of C1s, the EGF-homologous part, the two SCR units, and the two domains typical for C1s and C1r are localized within these domains.

The glycoprotein Ib/IX complex on platelets is responsible for the first stage of haemostasis as an essential component in the primary adhesion of platelets to damaged vessel walls. Glycocalicin is the extracellular part of platelet glycoprotein Ib alpha and contains the von Willebrand factor and thrombin binding sites. Disulphide bonds are implicated in the von Willebrand binding site and studies with peptides point towards a region of glycocalicin with four cysteines as containing the binding sites for both von Willebrand factor and thrombin. The position and linkage of these two disulphide bonds are now determined to be 209-248 and 211-264 and the relevance of this double-loop structure for glycoprotein Ib/IX function is discussed.

Secretory actin-binding protein (SABP), a glycoprotein from human seminal plasma, was isolated according to Akiyama and Kimura [Akiyama, K. & Kimura, H. (1990) Biochim. Biophys. Acta 1040, 206-210]. The complete amino acid sequence of SABP was determined with the aid of fragments generated by trypsin, Staphylococcus aureus V8 protease and pepsin. The single polypeptide chain of SABP contains 118 amino acids with a calculated Mr of 13,506 and pyroglutamic acid as the N-terminal residue. A single N-glycosidic carbohydrate moiety is located at Asn77. The carbohydrate composition shows an unusually high amount of fucose. The arrangement of the two disulfide bonds is Cys37-Cys63 and Cys61-Cys95. Sequence comparison revealed a high degree of similarity with a 14-kDa submandibular gland protein from mouse (45% identity and 64% similarity). SABP is identical with a prolactin-inducible protein and a protein termed gross cystic disease fluid protein 15 (sequences translated from cDNA clones), both from human breast tissues. Although SABP was also detected in saliva, in extracts of the submandibular gland and seminal vesicles, little is known of its function.

The structures of the N‐and O‐glycans of human, bovine and porcine plasminogen were determined by 500‐MHz 1H‐NMR spectroscopy. The N‐glycans of all three species proved to be of the N‐acetyllactosamine type differing from one another with respect to the sialylation and fucosylation patterns. In the N‐glycan of human plasminogen the two antennae are sialylated with N‐acetylneuraminic acid (NeuAc), whereas in the bovine counterpart both branches carry significant amounts of N‐glycolylneuraminic acid (NeuGc). In porcine plasminogen the sialic acid is mainly NeuAc; the Manα1→6 branch, however, is only partially sialylated. In addition, the porcine N‐glycan is fucosylated to about 80% in α1→6 linkage to the GlcNAc‐1 residue. The O‐glycans of the three species possess an identical Galβ1→3GalNAc core which is α2→3 sialylated with NeuAc at Gal. The disialylated form, which is also present in all three species, has an additional NeuAc residue in α2→6 linkage to GalNAc. Mono‐and disialylated forms occur in different molar ratios in the different plasminogens: 80:20 in human, 70:30 in bovine and 50:50 in porcine. This study on the carbohydrate moiety of these three plasminogens reveals species specificity in terms of various types of microheterogeneities.

The N-terminal amino acid sequence of equine, ovine, canine, goat and rabbit plasminogen were determined and compared with those already known of the human, bovine, porcine and feline molecule. Furthermore, the kringle 4 domains of equine, ovine, canine and goat plasminogen, prepared by limited cleavage with elastase, were sequenced and compared with the known species of human, bovine, porcine and chicken plasminogen. Homology with the human kringle 4 ranges between 73% (chicken) and 90% (bovine). Comparison of sequences, fragmentation patterns with elastase and adsorption on lysine-Bio-Gel suggests the same structural and functional domains in the animal species as in human plasminogen.

The amino acid sequence of the heavy chain of porcine plasmin (560 residues) was determined. Selected fragments were produced by cleavage with cyanogen bromide, elastase, Staphylococcus aureus V8 protease, trypsin or clostripain or with combinations thereof. The fragments were aligned with overlapping sequences. Sequence comparison with the human and bovine species indicated that the porcine molecule contains the same structural and functional domains as the two others. In particular, the characteristic arrangement of the disulfide bonds clearly indicates the five kringle structures. The heavy chain of porcine plasmin has a sequence homology of 84% with the bovine and of 78% with the human molecule. The average degree of identity among the kringles is over 80%. In the connecting strands between the kringles the identity decreases drastically (to 42% in the connecting strand between kringles 4 and 5) except for the strand between kringles 2 and 3 (over 80%).

gamma-Seminoprotein (gamma-SM), a glycoprotein from human seminal plasma, was isolated in highly purified form by ion-exchange chromatography on a Mono Q column. The main form, fraction M, was homogeneous by PAGE at pH 8.3 and by SDS-PAGE. The complete amino acid sequence of gamma-SM was determined with the aid of fragments generated by cleavages with cyanogen bromide, clostripain, chymotrypsin and Staphylococcus aureus V8 protease. The fragments were aligned with overlapping sequences. The single polypeptide chain of gamma-SM contains 237 amino acids with a calculated Mr of 26079. A single N-linked carbohydrate side chain is attached to Asn45. The complex structure of this oligosaccharide has been determined recently [van Halbeek H. et al. (1985) Biochem. Biophys. Res. Commun. 131, 507-514]. Sequence comparison with serine proteases shows a high degree of homology, especially with the kallikrein family. The residues in the vicinity of the active site of serine proteases are also highly conserved in gamma-SM, indicating the participation of His41, Asp96 and Ser189 in its active site. gamma-SM hydrolyzed M-casein with a pH optimum at 8.0, but failed to hydrolyze any of the synthetic substrates tested. This proteolytic activity could be inhibited with diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, Zn2+ or Hg2+ ions.

Normal human plasma alpha 2HS-glycoprotein has earlier been shown to be comprised of two polypeptide chains. Recently, the amino acid and carbohydrate sequences of the short chain were elucidated (Gejyo, F., Chang, J.-L., Bürgi, W., Schmid, K., Offner, G. D., Troxler, R.F., van Halbeck, H., Dorland, L., Gerwig, G. J., and Vliegenthart, J.F.G. (1983) J. Biol. Chem. 258, 4966-4971). In the present study, the amino acid sequence of the long chain of this protein, designated A-chain, was determined and found to consist of 282 amino acid residues. Twenty-four amino acid doublets were found; the most abundant of these are Pro-Pro and Ala-Ala which each occur five times. Of particular interest is the presence of three Gly-X-Pro and one Gly-Pro-X sequences that are characteristic of the repeating sequences of collagens. Chou-Fasman evaluation of the secondary structure suggested that the A-chain contains 29% alpha-helix, 24% beta-pleated sheet, and 26% reverse turns and, thus, approximately 80% of the polypeptide chain may display ordered structure. Four glycosylation sites were identified. The two N-glycosidic oligosaccharides were found in the center region (residues 138 and 158), whereas the two O-glycosidic heterosaccharides, both linked to threonine (residues 238 and 252), occur within the carboxyl-terminal region. The N-glycans are linked to Asn residues in beta-turns, while the O-glycans are located in short random segments. Comparison of the sequence of the amino- and carboxyl-terminal 30 residues with protein sequences in a data bank demonstrated that the A-chain is not significantly related to any known proteins. However, the proline-rich carboxyl-terminal region of the A-chain displays some sequence similarity to collagens and the collagen-like domains of complement subcomponent C1q.

The Schizosaccharomyces pombe acid phosphatase structural gene (PHO 1) was isolated by complementation of an S. pombe acid phosphatase mutant with a wild type S. pombe DNA recombinant plasmid library. Northern analysis indicates that acid phosphatase is encoded by a 1.4-kilobase mRNA of which approximately 100 bases are 3'-poly(A). The gene contains no introns and the 3' and 5' untranslated regions are short. According to DNA and amino acid sequence data, the S. pombe acid phosphatase has a molecular weight of 50,600. An 18-amino acid sequence at the N terminus was found that is similar to previously identified signal peptides in other eukaryotic secretory proteins. This signal peptide is apparently removed during secretion, since it is absent in the mature secreted acid phosphatase. The gene can be induced 2--3-fold by starvation for phosphate. The signals required for this induction are contained on the isolated DNA clone. Although the gene can be expressed in Saccharomyces cerevisiae, secretion is abnormal.

Human C1s proenzyme (Mr 83 000) was isolated by a rapid two-stage method involving affinity chromatography of C1 on IgG-Sepharose and isolation of subcomponent C1s by ion-exchange chromatography on DEAE-Sephacel. Single-chain C1s proenzyme was activated to two-chain C1s with self-activated C1r. After reduction and S-carboxamidomethylation the heavy chain of C1s (Mr 57 000) was isolated by ion exchange chromatography on DEAE-Sephacel. Cleavage of C1s heavy chain with CNBr yielded five fragments whose N-terminal sequences were determined. The alignment of the fragments within the heavy chain was established by tryptic peptides containing methionine. C1s heavy chain comprises about 470 amino acid residues and 42% of its sequence was determined. An intrachain sequence homology and a homology to the alpha 2 chain of human haptoglobin were identified. The C-terminal CNBr fragment comprising 44 amino acid residues was completely sequenced. From BNPS-skatole cleavage of reduced and alkylated C1s proenzyme a fragment was isolated which overlaps the C1s heavy and light chain parts and which contains the peptide bond cleaved during activation. The results show that this is an Arg-Ile bond and that under standard conditions of activation no peptide material is liberated from this portion of the molecule. The sequence data and homology to two-chain serine proteases indicate a single interchain disulfide bond in C1s.

The complete amino acid sequence of porcine miniplasminogen (Mr 37 600), comprising 341 residues, was determined by automated Edman degradation in a liquid-phase or solid-phase sequenator. Selected fragments were produced by cleavage with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine (BNPS-skatole), cyanogen bromide, hydroxylamine, Staphylococcus aureus protease or trypsin or with combinations thereof and by activation with urokinase. The sequence obtained was compared with the known sequences of human and bovine miniplasminogen, indicating that the porcine molecule apparently contains the same structural and functional domains as the protein of the other two species. Porcine miniplasminogen has a sequence homology of 83% with human and of 79% with bovine miniplasminogen; 74% of the amino acids are identical in all three species. The results show a higher degree of evolutionary conservatism in the structurally and/or functionally vital regions of the molecule (active site residues, kringle 5).

The amino acid sequence of the single polypeptide chain of bovine plasminogen (786 residues, Mr 88092) was determined. Cleavage with CNBr yielded 13 fragments of which six originated from cleavage sites different from human plasminogen. Digestion with elastase gave three major fragments: kringles (1 + 2 + 3) and kringle 4, both with intact lysine binding sites, and mini-plasminogen. Subfragmentation was achieved mainly with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine (BNPS-skatole), Staphylococcus aureus V8 protease and trypsin. The sequences of fragments which were determined by automated Edman degradation, were aligned with overlapping sequences, or, in a few instances, by homology with the known sequence of human plasminogen. Sequence comparison with the human protein showed varying degrees of homology in the different functional and structural domains. The overall identity (78%) is practically the same as that found in those regions corresponding to the heavy (79%) and the light chain (80%) of plasmin. The average degree of identity among the kringles is 83%. Outside the kringle structures the extent of identity decreases, to 65% in the N-terminal region and to about 50% in the connecting strands between the kringles except for the strand between kringles 2 and 3, where only one out of 12 residues is exchanged. The results reported show that bovine plasminogen apparently contains the same structural and functional domains as human plasminogen. Bovine plasminogen also contains two carbohydrate moieties. The only partially substituted N-glycosidic site, Asn289, corresponds to partially glycosylated Asn288 in human plasminogen, whereas the O-glycosidic site of the human sequence, Thr345, is shifted to Ser339 in bovine plasminogen.