The catalytic chain of human complement subcomponent C1r. Purification and N-terminal amino acid sequences of the major cyanogen bromide-cleavage fragments.

1. The a- and b-chains of reduced and alkylated human complement subcomponent C1r were separated by high-pressure gel-permeation chromatography and isolated in good yield and in pure form. 2. CNBr cleavage of C1r b-chain yielded eight major peptides, which were purified by gel filtration and high-pressure reversed-phase chromatography. As determined from the sum of their amino acid compositions, these peptides accounted for a minimum molecular weight of 28 000, close to the value 29 100 calculated from the whole b-chain. 3. N-Terminal sequence determinations of C1r b-chain and its CNBr-cleavage peptides allowed the identification of about two-thirds of the amino acids of C1r b-chain. From our results, and on the basis of homology with other serine proteinases, an alignment of the eight CNBr-cleavage peptides from C1r b-chain is proposed. 4. The residues forming the 'charge-relay' system of the active site of serine proteinases (His-57, Asp-102 and Ser-195 in the chymotrypsinogen numbering) are found in the corresponding regions of C1r b-chain, and the amino acid sequence around these residues has been determined. 5. The N-terminal sequence of C1r b-chain has been extended to residue 60 and reveals that C1r b-chain lacks the 'histidine loop', a disulphide bond that is present in all other known serine proteinases.     

A collagen-like amino acid sequence in a polypeptide chain of human C1q (a subcomponent of the first component of complement)

1. A partial amino acid sequence of 95 residues of the 191 residues in the oxidized A chain of human subcomponent C1q was determined. The partial nature of the sequence is because one overlapping peptide is missing in the proposed sequence, also the proof of some of the overlapping peptides depends partly on their amino acid composition and not on their complete sequence. 2. This region of the A chain contained a repeating sequence of glycine-X-Y (where X is often proline and Y is often hydroxyproline) for 78 residues. 3. The five hydroxylysine residues and the five hydroxyproline residues present in the oxidized A chain were all in these 78 residues and only in the Y position of the repeating sequence. 4. Prolonged collagenase digestion of the oxidized A chain yielded a large, apparently C-terminal, peptide which contained most of the non-collagenous sequences present in the chain. 5. It is concluded that there is a collagen-like region in the A chain of subcomponent C1q which constitutes most of the N-terminal half of the chain and that similar collagen-like regions will be found in the B and C chains.     

Amino acid sequence of the N-terminal 108 amino acid residues of the B chain of subcomponent C1q of the first component of human complement.

The amino acid sequence of the N-terminal 108 residues of the B chain of subcomponent C1q of the first component of human complement was determined. The B chain has a blocked N-terminal amino acid, which was judged to be 5-oxopyrrolidine-2-carboxylic acid. A collagen-like region of 84 residues was found, which started at position B-6, and all of the six hydroxylysine residues and 12 hydroxyproline residues present in the chain were found in this region. Four of the six hydroxylysine residues may be glycosylated. The repeating nature of the collagen-like region is broken at position B-9, where alanine is found in a position where glycine would be expected. The exact position of the interchain disulphide bond joining the A and B chains of human subcomponent C1q was shown to be between residues A4 and B4.     

Complete amino acid sequence of the A chain of human complement-classical-pathway enzyme C1r.

The amino acid sequence of human C1r A chain was determined, from sequence analysis performed on fragments obtained from C1r autolytic cleavage, cleavage of methionyl bonds, tryptic cleavages at arginine and lysine residues, and cleavages by staphylococcal proteinase. The polypeptide chain has an N-terminal serine residue and contains 446 amino acid residues (Mr 51,200). The sequence data allow chemical characterization of fragments alpha (positions 1-211), beta (positions 212-279) and gamma (positions 280-446) yielded from C1r autolytic cleavage, and identification of the two major cleavage sites generating these fragments. Position 150 of C1r A chain is occupied by a modified amino acid residue that, upon acid hydrolysis, yields erythro-beta-hydroxyaspartic acid, and that is located in a sequence homologous to the beta-hydroxyaspartic acid-containing regions of Factor IX, Factor X, protein C and protein Z. Sequence comparison reveals internal homology between two segments (positions 10-78 and 186-257). Two carbohydrate moieties are attached to the polypeptide chain, both via asparagine residues at positions 108 and 204. Combined with the previously determined sequence of C1r B chain [Arlaud & Gagnon (1983) Biochemistry 22, 1758-1764], these data give the complete sequence of human C1r.     

Complete amino acid sequences of the three collagen-like regions present in subcomponent C1q of the first component of human complement.

Possible interactions between polymerized (F-) actin and insulin-storage granules from rat islets of Langerhans were examined in vitro by comparing the sedimentation of the granules in the presence of various actin concentrations. Actin in the concentration range 0.1--0.5 mg/ml produced a retardation in granule-sedimentation rates consistent with binding of the granules to the actin filaments. The interaction was increased by addition of ATP (2mM), but was decreased by CaCl2 (0.1 mM). Binding of granules to actin was unaffected by cyclic AMP or by preincubation of the granules with phospholipase C. Specificity of the interaction was confirmed by the use of depolymerized (G-) actin and of myosin to provide a solution of comparable viscosity; neither of these caused any alteration of granule sedimentation. Possible implications of this interaction of insulin-storage granules with actin for the mechanism of insulin secretion are briefly discussed.     

Complete amino acid sequence of human alpha 1-microglobulin

Complete amino acid sequence of human α₁-microglobulin has been established. It is composed of 167 amino acid residues and contains three carbohydrate attachment sites. No amino acid sequence heterogeneity was found.     

Functional model of subcomponent C1 of human complement

The domain organization of the zymogen subunits of the first component of human complement C1s, C1r2 and the complex C1s-C1r2-C1s was studied by electron microscopy. In the absence of Ca2+, monomeric C1s was visualized as a dumb-bell-shaped molecule consisting of two globular domains (center-to-center distance 11 nm) connected by a rod. One of the globular domains is assigned to the light chain (B-chain) of the activated molecule, which is homologous to trypsin and other serine proteases. The second globular domain and the rod are assigned to the heavy chain (A-chain) of CIs. The subunit C1r is a stable dimer in the presence or absence of Ca2+. This dimer C1r2 was visualized as composed of two dumb-bells of dimensions similar to those observed for C1s. These are connected near the junctions between the rod and one of the globular domains. This leads to the structure of an asymmetrical X with two inner closely spaced globules (center-to-center distance 7 nm) and two outer globules at a larger distance (14 nm). By comparison with fragment C1rII2, in which part of the A-chain is removed, the inner globular domains were assigned to the catalytic B-chains. This characteristic structure of C1r2 is readily recognized in the central portion of the thread-like 54 nm long C1s-C1r2-C1s complex formed in the presence of Ca2+. By affinity-labeling of C1s with biotin and visualization of avidin-ferritin conjugates in the reconstituted complex, it was demonstrated that C1s forms the outer portion of the complex. A detailed model of C1s-C1r2-C1s is proposed, according to which two C1s monomers bind to the outer globes of C1r2 by contacts between their heavy chains and those of C1r. According to this model the catalytic domains of C1r are located in the center and those of C1s at the very tips of the C1s-C1r2-C1s complex. On the basis of the structure of C1s-C1r2-C1s, we derived a detailed model of the C1 complex (composed of C1q and the tetrameric complex) and we discuss this model with a view to finding a possible activation mechanism of C1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Complete amino acid sequence of human serum cholinesterase

The complete amino acid sequence of human serum cholinesterase (choline esterase II (unspecific), EC 3.1.1.8) was determined by Edman degradation of purified peptides. The protein contains 574 amino acids per subunit and nine carbohydrate chains attached to 9 asparagines. The four subunits of cholinesterase appear to be identical. The active site serine is the 198th residue from the amino terminus. The sequence of human serum cholinesterase is 53.8% identical with the sequence of acetylcholinesterase from Torpedo californica and 28% identical with the carboxyl-terminal portion of bovine thyroglobulin.     

Complete amino acid sequence of a human platelet proteoglycan

The primary structure of a human platelet proteoglycan (P.PG) core was established by a combination of amino acid sequence analysis and cDNA cloning. The deduced 131 amino acid long protein contains eight Ser-Gly repeats. The significance of homologies observed between P.PG and promyelocytic leukemia cell line proteoglycans is discussed.     

Molecular cloning of cDNA for human complement component C1s. The complete amino acid sequence

The complete amino acid sequence (673 residues plus 15 residues of leader sequence) of human complement component C1s has been determined by nucleotide sequencing of cDNA clones from a human liver library probed with synthetic oligonucleotides. Much of the sequence is supported by independent amino acid sequence information. The cDNA sequence contains an anomalous "intron-like" sequence, including a stop codon, that can be discounted because of the amino acid sequence evidence. The N-terminal chain (422 residues) of C1s, like that of C1r with which it is broadly homologous, contains five domains: domains I and III are homologous to one another and to similar regions in C1r, domain II is homologous to the epidermal growth factor sequence found in C1r and several other proteins, and domains IV and V are homologous to one another and to the 60-residue repeating sequence found in C1r, C2, factor B, C4-binding protein and some apparently unrelated proteins. The sequence of the C-terminal chain (251 residues) agrees with that already established to be the "serine protease" domain of C1s.     

Completion of the amino acid sequences of the A and B chains of subcomponent C1q of the first component of human complement.

The sequences of amino acid residues 109--224 of the A chain, and residues 109--22 of the B chain, of human subcomponent C1q are given. These results, along with previously published sequence data on the N-terminal, collagen-like, regions of the A and B chains [Reid (1979) Biochem. J. 179, 367--371] yield the complete amino acid sequences of the A and B chains of subcomponent C1q. The asparagine residue at position A-124 has been identified as the major site of asparagine-linked carbohydrate in subcomponent C1q. When the sequences of the C-terminal, 135-residue-long, 'globular' regions of A and B chains are compared they show 40% homology. The degree of homology over certain stretches of 15--20 residues, within the C-terminal regions, rises up to values of 73%, indicating the presence of strongly conserved structures. Structure prediction studies indicate that both the A and B chain C-terminal regions may adopt a predominantly beta-type structure with apparently little alpha-helical structure.     

Autocatalytic activation of C1r subcomponent of the first component of human complement

Autoactivation of the proenzyme form of a subunit of the first component (C1r) was performed in the presence and absence of diisopropyl fluorophosphate (DFP). The time-course of autoactivation of zymogen C1r followed a sigmoidal curve and was accelerated by addition of the enzyme C1r and by increasing the concentration of C1r, suggesting that autoactivation of C1r consists of two intermolecular reactions, i.e. zymogen(C1r)- and enzyme(C1r)-catalyzed reactions. In the presence of 10 mM DFP, the enzyme-catalyzed autoactivation of C1r was completely inhibited, while the zymogen-catalyzed autoactivation still proceeded depending upon C1r concentration. These results suggested that the zymogen-catalyzed autoactivation of C1r is a DFP-insensitive second-order reaction and is mediated by an active site generated in a single chain C1r through a conformational change (Kassahara et al. (1982) FEBS lett. 141, 128-131). Based on these results, a possible reaction process of autoactivation of C1r was proposed, as follows: (formula; see text) where C1r represents a conformational isomer which catalyzes the autoactivation of C1r, and the rate constants, k2 and k3, are of second-order. Utilizing a computer, we simulated the autoactivation of C1r and found the above scheme to be a reasonable model of C1r autoactivation. Evidence which supports the formation of a conformational isomer of C1r, C1r, as an intermediate in its autoactivation was also obtained by a surface radiolabeling method.     

Complete amino acid sequence of human phosphoglycerate kinase. Isolation and amino acid sequence of tryptic peptides

As a part of the elucidation of the complete amino acid sequence of human phosphoglycerate kinase, 46 tryptic peptides, ranging in length from 1 to 26 residues, were isolated and characterized from the reduced and S-carboxymethylated enzyme. The isolated peptides were subjected to sequence analysis by the modified dansyl-Edman degradation procedure and automated Edman degradation technique. The results, together with the data on cyanogen bromide peptides and two additional tryptic peptides from cyanogen bromide peptides reported in the accompanying paper, established the complete amino acid sequence of human erythrocyte phosphoglycerate kinase.     

Calcium-sensitive thermal transitions and domain structure of human complement subcomponent C1r

Fluorescent probes and other methods have been used to investigate the thermal stability of activated C1r and functionally intact fragments isolated from tryptic digests of the protein. This enzyme exhibits two irreversible transitions that differ with respect to their sensitivity to metal ions. The high-temperature transition occurs with a midpoint near 53 degrees C in 0.02 M tris(hydroxymethyl)aminomethane buffer and 0.15 M NaCl, pH 7.4. It is relatively insensitive to Ca2+ and ionic strength and is accompanied by a loss of catalytic activity. The low-temperature transition is most easily observed in the presence of ethylenediaminetetraacetic acid and is completely abolished by 100 microM Ca2+. Its midpoint varies between 26 degrees C at low ionic strength and 40 degrees C in the presence of 0.5 M NaCl. The low-temperature transition results in extensive polymerization of the protein without loss of the esterolytic activity or the ability to react with C1 inhibitor; however, the ability to reconstitute hemolytically active C1 or even bind to C1s in the presence of Ca2+ is destroyed. A highly purified N-terminal fragment generated by tryptic digestion of C1r in the presence of Ca2+ retained its ability to interact with C1s, disrupting the formation of C1s dimers in the presence of Ca2+. In the absence of Ca2+, this fragment displays only a low-temperature transition that is very similar to the one observed with the whole protein and that destroys its ability to bind to C1s. Addition of Ca2+ stabilizes this fragment, shifting the midpoint of its melting transition upward by more than 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydroxylysine-linked glycosides of human complement subcomponent C1q and various collagens.

1. Human C1q, a subcomponent of the first component of complement, contains 67 disaccharides (glucosylgalactose) and 2.4 monosaccharides (galactose) linked to hydroxylysine in one molecule. It was found that 82.6% of the hydroxylsine residues were glycosylated. The suggestion of the possible existence of glucosylgalactosylhydroxylysine reported previously [Yonemasu, Stroud, Niedermeir & Butler (1971) Biochem. Biophys. Res. Commun. 43, 1388--1394] was confirmed. 2. The hydroxylysine-glycosides are not detected in the C-terminal, non-collagen-like, globular regions, but only in the collagen-like regions in the subcomponent C1q molecule. 3. Alpha 1(I) and alpha 2 in pig skin, alpha 1(II) in bovine cartilage and alpha 1(III) in bovine skin collagens contain 2.0, 2.2, 13.2 and 2.0 residues of hydroxylysine-glycosides per molecule, respectively. The percentage of hydroxylysine residues glycosylated in each of these chains is relatively low (on average 38%). 4. Neither the high percentage of hydroxylysine residues glycosylated nor the high values for the ratios of disaccharides to monosaccharides in the subcomponent C1q resembles that in alpha 1(I), alpha 2, alpha 1(II) and alpha 1(III). 5. Similarities between the extent of glycosylation of hydroxylysine residues in collagen-like regions in the subcomponent C1q molecule and that of the collagenous constituents of human glomerular basement membranes, aortic intima, skin A- and B-chains and of bovine anterior lens capsule are discussed.     

The complete amino acid sequence of human complement factor H.

The complete amino acid sequence of the human complement system regulatory protein, factor H, has been derived from sequencing three overlapping cDNA clones. The sequence consists of 1213 amino acids arranged in 20 homologous units, each about 60 amino acids long, and an 18-residue leader sequence. The 60-amino-acid-long repetitive units are homologous with those found in a large number of other complement and non-complement proteins. Two basic C-terminal residues, deduced from the cDNA sequence, are absent from factor H isolated from outdated plasma. A tyrosine/histidine polymorphism was observed within the seventh homologous repeat unit of factor H. This is likely to represent a difference between the two major allelic variants of factor H. The nature of the cDNA clones indicates that there is likely to be an alternative splicing mechanism, resulting in the formation of at least two species of factor H mRNA.     

The amino acid sequence of a human κ light chain

The amino acid sequence of the light chain of the myeloma protein Dee was studied. The light chain is of the kappa type and of subgroup I. The variable part contains some substitutions that are unique and also some that have been observed already in other kappaI chains (repeated variants). Based on these repeated variants a subdivision of the kappaI subgroup is proposed.     

Complete amino acid sequence of a beta-galactoside-binding lectin from human placenta

The complete amino acid sequence of a beta-galactoside-binding lectin from human placenta was determined at protein level. The lectin consists of 134 amino acids and its N-terminal alanine is blocked with acetate. The lectin shows about 50% similarity with chick 14K lectin, which was the first vertebrate beta-galactoside-binding lectin completely sequenced. Only 14 residues proved to be different from those of rat lung lectin, the sole mammalian lectin of which the complete sequence has been reported.