The serine proteinase chain of human complement component C̅1s. Cyanogen bromide cleavage and <i>N</i>-terminal sequences of the fragments

Carter, P E; Dunbar, Bryan; Fothergill, John E.

doi:10.1042/bj2150565

Cited by 26 publications

(11 citation statements)

References 24 publications

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“…In this respect, it should be mentioned that plasmin degradation of the serine protease domain of C1 s was reported to result in the loss of C4 binding ability (14). In the same way, it has been suggested that C1 s may interact with the C4a moiety of C4 through acidic residues close to the active site (41). It may be hypothesized, therefore, that efficient positioning of C4 with respect to C1 s requires two types of interactions: (i) between the C4b moiety and the CCP modules and (ii) between the C4a moiety and the serine protease domain.…”

Section: Discussionmentioning

confidence: 99%

Baculovirus-mediated Expression of Truncated Modular Fragments from the Catalytic Region of Human Complement Serine Protease C1s

Rossi

Bally

Thielens

et al. 1998

Journal of Biological Chemistry

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C1s is the modular serine protease responsible for cleavage of C4 and C2, the protein substrates of the first component of complement. Its catalytic region (␥-B) comprises two complement control protein (CCP) modules, a short activation peptide (ap), and a serine protease domain (SP). A baculovirus-mediated expression system was used to produce recombinant truncated fragments from this region, deleted either from the first CCP module (CCP 2 -ap-SP) or from both CCP modules (ap-SP). The aglycosylated fragment CCP 2 -ap-SPag was also expressed by using tunicamycin. The fragments were produced at yields of 0.6 -3 mg/liter of culture, isolated, and characterized chemically and then tested functionally by comparison with intact C1s and its proteolytic ␥-B fragment. All recombinant fragments were expressed in a proenzyme form and cleaved by C1 r to generate active enzymes expressing esterolytic activity and reactivity toward C1 inhibitor comparable to those of intact C1 s. Likewise, the activated fragments ␥-B, CCP 2 -ap-SP, and ap-SP retained C1 s ability to cleave C2 in the fluid phase. In contrast, whereas fragment ␥-B cleaved C4 as efficiently as C1 s, the C4-cleaving activity of CCP 2 -ap-SP was greatly reduced (about 70-fold) and that of ap-SP was abolished. It is concluded that C4 cleavage involves substrate recognition sites located in both CCP modules of C1 s, whereas C2 cleavage is affected mainly by the serine protease domain. Evidence is also provided that the carbohydrate moiety linked to the second CCP module of C1 s has no significant effect on catalytic activity.The first component of complement comprises two homologous, yet distinct modular serine proteases C1r 1 and C1s assembled in the form of a Ca 2ϩ -dependent tetramer C1s-C1r-C1r-C1s. C1r is responsible for intrinsic C1 activation, a twostep process first involving autolytic C1r activation and then C1 r-mediated cleavage of proenzyme C1s. The active enzyme C1 s is a highly specific protease with trypsin-like specificity that mediates limited proteolysis of C4 and C2, the two protein substrates of C1 , thereby triggering activation of the classical pathway of complement in response to infection by various microorganisms (2-5). C4 cleavage occurs in the fluid phase and generates fragment C4b, which has the transient ability to bind covalently to the surface of the target. Cleavage of the second substrate C2, in contrast, takes place after prior formation of a C4b-C2 complex and yields C4b-C2a, the protease responsible for cleavage of complement protein C3 (5). This double proteolytic activity of C1 is controlled by C1 inhibitor, a member of the serine protease inhibitor (serpin) family, which reacts stoichiometrically with both C1 r and C1 s to form covalent protease-inhibitor complexes, resulting in disruption of the C1 s-C1 r-C1 r-C1 s tetramer (2).Human C1s is synthesized as a 673-residue single chain zymogen which, upon activation by C1 r, is cleaved between Arg 422 and Ile 423 to yield two disulfide-linked polypeptides, the N-terminal A chain ...

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Section: Discussionmentioning

confidence: 99%

Baculovirus-mediated Expression of Truncated Modular Fragments from the Catalytic Region of Human Complement Serine Protease C1s

Rossi

Bally

Thielens

et al. 1998

Journal of Biological Chemistry

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“…The phenylthiohydantoin samples were analysed by chromatography on a Waters Resolve C18 reverse-phase column with a pH 5.0 acetate/acetonitrile buffer system (Carter et al, 1983).…”

Section: Automatic Amino Acid Sequence Determinationmentioning

confidence: 99%

The cloning and expression of the aroL gene from Escherichia coli K12. Purification and complete amino acid sequence of shikimate kinase II, the aroL-gene product

Millar¹,

Lewendon²,

Hunter³

et al. 1986

Biochemical Journal

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The aroL gene encoding the enzyme shikimate kinase II was cloned from Escherichia coli K12. Construction of over-expressing strains permitted for the first time the purification to homogeneity of a monofunctional shikimate kinase. The complete amino acid sequence of shikimate kinase II was determined by a combined nucleotide and direct amino acid sequencing strategy. E. coli shikimate kinase II is a monomeric enzyme containing 173 amino acid residues with a calculated Mr 18 937. The amino acid sequence contains a region homologous with other kinases and ATP-requiring enzymes. Evidence is presented suggesting that the transcriptional start site of the aroL gene is located within a potential operator site.

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“…This was performed on a TSK 2000 SW column with either 0.3 M-sodium phosphate, pH 6.9, or 0.08 M-sodium phosphate/0.32 M-NaCl/20% (v/v) Smith et al (1982) and Carter et al (1983).…”

Section: Materials and Methods Materialsmentioning

confidence: 99%

Physicochemical and immunochemical characterization of allergenic proteins from rye-grass (Lolium perenne) pollen prepared by a rapid and efficient purification method

Cottam¹,

Moran²,

Standring³

1986

Biochemical Journal

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Three fractions of rye-grass (Lolium perenne) pollen extract have been isolated by preparative isoelectric focusing (i.e.f.) and characterized in terms of physicochemical and immunochemical properties. The purified components were designated 'R7' and 'R14' on the basis of their positions in relation to other rye-grass pollen extract components on SDS/polyacrylamide-gel electrophoresis and their apparent molecular masses were assessed as 31 and 11 kDa respectively. On i.e.f., R14 split into two components, one acidic (pl 5.0) and one basic (pl 9.0), termed 'Rl4a' and 'Rl4b' respectively, and R7 focused at pl 5.8. R7 and R14a were shown to be allergenic by skin-prick test and all three components were recognized by rye-grass-pollen-specific human IgE. On SDS/polyacrylamide-gel electrophoresis and i.e.f., R7 behaved in a manner identical with that shown by an authentic sample of Rye I and gave an amino acid analysis similar to published data Immunochemistry 3, 91-100] for Rye group-I isoallergens; the amino acid sequence of the first 27 N-terminal amino acids was also determined. Physicochemical analysis revealed that R14a was equivalent to Rye II and 14b to Rye III. Preparative i.e.f. followed by gel-permeation chromatography proved to be a rapid and efficient method for purifying the allergenic components of Rye I (R7), Rye II (Rl4a) and Rye III (Rl4b) from rye-grass pollen extract.

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The serine proteinase chain of human complement component C̅1s. Cyanogen bromide cleavage and N-terminal sequences of the fragments

Cited by 26 publications

References 24 publications

Baculovirus-mediated Expression of Truncated Modular Fragments from the Catalytic Region of Human Complement Serine Protease C1s

Baculovirus-mediated Expression of Truncated Modular Fragments from the Catalytic Region of Human Complement Serine Protease C1s

The cloning and expression of the aroL gene from Escherichia coli K12. Purification and complete amino acid sequence of shikimate kinase II, the aroL-gene product

Physicochemical and immunochemical characterization of allergenic proteins from rye-grass (Lolium perenne) pollen prepared by a rapid and efficient purification method

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