Analysis of the N‐linked oligosaccharides of human C1s using electrospray ionisation mass spectrometry

Pétillot, Yves; Thibault, Pierre; Thielens, Nicole M.; Rossi, Véronique; Lacroix, Monique; Coddeville, Bernadette; Spik, Geneviève; Schumaker, Verne N.; Gagnon, Jean; Arlaud, Gérard J.

doi:10.1016/0014-5793(94)01429-5

Cited by 27 publications

(14 citation statements)

References 23 publications

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“…Purified human ␣ 2 -macroglobulin (␣ 2 M) was kindly provided by L. Sottrup-Jensen (University of Aarhus). The concentrations of purified proteins were determined using the following absorption coefficients (A 1 cm 1% at 280 nm) and molecular weights: C1s, 14.5 and 79,800; C4, 8.3 and 205,000; C2, 8.9 and 102,000; C3, 10.0 and 185,000 (29,30,33). The absorption coefficients (A 1 cm 1% at 280 nm) used for the recombinant fragments CCP1/2-SP and CCP2-SP of MASP-2 (18.3 and 19.2, respectively) were calculated from the number of Trp, Tyr, and disulfides according to the method of Edelhoch (34), using molecular weight values of 42,710 and 35,330, calculated from the sequence (6).…”

Section: Methodsmentioning

confidence: 99%

Substrate Specificities of Recombinant Mannan-binding Lectin-associated Serine Proteases-1 and -2

Rossi¹,

Cseh²,

Bally³

et al. 2001

Journal of Biological Chemistry

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Mannan-binding lectin (MBL)-associated serine proteases-1 and 2 (MASP-1 and MASP-2) are homologous modular proteases that each interact with MBL, an oligomeric serum lectin involved in innate immunity. To precisely determine their substrate specificity, human MASP-1 and MASP-2, and fragments from their catalytic regions were expressed using a baculovirus/insect cells system. Recombinant MASP-2 displayed a rather wide, C1s-like esterolytic activity, and specifically cleaved complement proteins C2 and C4, with relative efficiencies 3-and 23-fold higher, respectively, than human C1s. MASP-2 also showed very weak C3 cleaving activity. Recombinant MASP-1 had a lower and more restricted esterolytic activity. It showed marginal activity toward C2 and C3, and no activity on C4. The enzymic activity of both MASP-1 and MASP-2 was specifically titrated by C1 inhibitor, and abolished at a 1:1 C1 inhibitor:protease ratio. Taken together with previous findings, these and other data strongly support the hypothesis that MASP-2 is the protease that, in association with MBL, triggers complement activation via the MBL pathway, through combined self-activation and proteolytic properties devoted to C1r and C1s in the C1 complex. In view of the very low activity of MASP-1 on C3 and C2, our data raise questions about the implication of this protease in complement activation.Mannan-binding lectin (MBL) 1 is an oligomeric C-type lectin that recognizes arrays of neutral carbohydrates such as mannose and N-acetylglucosamine on the surface of pathogenic microorganisms (2). This selectivity endows MBL with the ability to discriminate self from infectious non-self, and confers this "ante-antibody" a major role in innate immunity, as underlined by numerous clinical reports indicating that MBL deficiency is linked with increased susceptibility to infectious diseases (3-5). In addition to its role as an opsonin (3), MBL has devised the ability to associate to several modular proteases termed MASPs (MBL-associated serine proteases) (6 -9). A single MASP entity was initially identified, and characterized as a protease with the ability to cleave complement proteins C4, C2, and C3 (7, 10). Further studies by Thiel et al. (6) revealed that MASP was indeed a mixture of two related but distinct proteases, MASP-1 and MASP-2, and that only the latter had the ability to cleave C4. A third protein component MAp19, arising from alternative splicing of the MASP-2 gene (11, 12), and very recently a further protease MASP-3 (13) were also shown to be associated with MBL.MASP-1 and MASP-2 show a domain organization identical to that of C1r and C1s, the enzymatic components of the C1 complex of complement (14), with an N-terminal CUB module (15) followed by an epidermal growth factor-like module, a second CUB module, two contiguous CCP modules (16), and a C-terminal chymotrypsin-like serine protease domain (see Fig. 1). By analogy with human C1s, it may be anticipated that the proteolytic activity and specificity of the MASPs is defined by the two CCP modules ...

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Section: Methodsmentioning

confidence: 99%

Substrate Specificities of Recombinant Mannan-binding Lectin-associated Serine Proteases-1 and -2

Rossi¹,

Cseh²,

Bally³

et al. 2001

Journal of Biological Chemistry

Self Cite

167

135

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“…The C1 s ␥-B fragment was obtained by limited proteolysis with plasmin (17) and purified by ion-exchange chromatography on a Mono Q HR 5/5 column (Pharmacia Biotech Inc.) as described previously (12). The two glycoforms of the C1 s ␥-B fragment, containing either a biantennary or a triantennary oligosaccharide, were isolated by affinity chromatography on concanavalin A-Sepharose (Pharmacia) as described previously (11). The desialylated C1 s ␥-B fragment was obtained by treatment with neuraminidase (10 units/mg protein) for 5 h at 25°C.…”

Section: Methodsmentioning

confidence: 99%

“…Complement proteins C2, C4, and C1 inhibitor were isolated from human plasma by means of published procedures (18 -20). For the determination of protein concentrations the following absorption coefficients (A (1%, 1 cm) at 280 nm) and molecular weights were used: C1r, 12.4 and 86,300 (21); C1s, 14.5 and 79,800 (21, 11); C1 s ␥-B, 18.3 and 47,520 (22,11); C2, 10.0 and 100,000 (18); C4, 8.2 and 205,000 (23,24); C1 inhibitor, 3.86 and 105,000 (25). The absorption coefficients (A (1%, 1 cm) at 280 nm) used for the recombinant fragments ap-SP (17.0) and CCP 2 -ap-SP (16.4) were calculated from their amino acid composition by the method of Edelhoch (26), and their molecular weights were determined by mass spectrometry analysis (see "Results").…”

Section: Methodsmentioning

confidence: 99%

“…The CCP modules (originally known as short consensus repeats) are structural motifs of about 60 residues homologous to those mostly found in various complement regulatory proteins known to interact with components C3b and/or C4b (10). The C-terminal CCP module of C1s bears a heterogeneous, complex-type N-linked oligosaccharide with both biantennary and triantennary species (11). Based on chemical cross-linking and three-dimensional homology modeling, it was shown recently that this module closely interacts with the serine protease domain on the side opposite to both the active site and the Arg 422 -Ile 423 bond cleaved upon activation (12).…”

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confidence: 99%

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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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“…Mass spectrometry analyses were performed by the electrospray ionization technique on an API III triple-quadrupole mass spectrometer (PE/Sciex, Thornill, Canada) equipped with a nebulizer-assisted electrospray (ionspray) source, as described previously [7].…”

Section: Eleetrospray Mass Spectrometrymentioning

confidence: 99%

NMR structures of a mitochondrial transit peptide from the green alga Chlamydomonas reinhardtii

et al. 1996

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The 26-amino-acid pre-sequence of the ATP synthase subunit that directs the protein from the cytosol to mitochondria in the unicellular green alga Chlamydomonas reinhardtii has been synthesised and analysed using NMR spectroscopy/circular dichroism and compared to a chloroplast transit peptide from the same organism. The results demonstrate that the peptide, though mainly unstructured in water, undergoes a strong conformational change in a 36% water/64% 2,2,2-trifluoroethanol mixture. In this solvent condition, an ~-helix was characterised by NMR from residue 2 to 26. Structure calculations under NMR restraints lead to a population of models of which 60% are kinked at position 9-10. Structural analysis indicates two hydrophobic sectors on the models with a discontinuity at the 9-10 kink level. The structures suggest a different interaction mode with the mitochondrial membrane compared to the chloroplast transit peptide.

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Analysis of the N‐linked oligosaccharides of human C1s using electrospray ionisation mass spectrometry

Cited by 27 publications

References 23 publications

Substrate Specificities of Recombinant Mannan-binding Lectin-associated Serine Proteases-1 and -2

Substrate Specificities of Recombinant Mannan-binding Lectin-associated Serine Proteases-1 and -2

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

NMR structures of a mitochondrial transit peptide from the green alga Chlamydomonas reinhardtii

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