Differential elution of Clq, Cl̄r and Cl̄s from human CT bound to immune aggregates. use in the rapid purification of Cl̄ sub-components

Arlaud, Gérard J.; Sim, Robert B.; Duplaa, A.M.; Colomb, Maurice G.

doi:10.1016/0161-5890(79)90069-5

Cited by 143 publications

(86 citation statements)

References 27 publications

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“…In the case of MASP-2, preincubation of the protease with 2 mM DFP for 30 min at 37°C resulted in 86% inhibition of its C4 cleaving ability, and nearly complete inhibition was obtained when this treatment was performed twice in a row. Thus, MASP-2 exhibited a DFP sensitivity comparable with that previously determined in the case of C1r and C1s (42). We also checked the effect of ␣ 2 M on the C4 cleaving activity of MASP-2 and C1s.…”

Section: Production and Characterization Of Recombinant Masp-2 And C-mentioning

confidence: 66%

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

Rossi¹,

Cseh²,

Bally³

et al. 2001

Journal of Biological Chemistry

162

135

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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: Production and Characterization Of Recombinant Masp-2 And C-mentioning

confidence: 66%

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

Rossi¹,

Cseh²,

Bally³

et al. 2001

Journal of Biological Chemistry

162

135

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“…Preparation and Analysis of the C1q Globular Domain-C1q was purified from human serum as described previously (22) and digested with Achromobacter iophagus collagenase (Roche Applied Science) (enzyme/protein ratio ϭ 0.2, w/w) for 24 h at 37°C in 250 mM NaCl, 5 mM * This work was supported by the Commissariat à l'Energie Atomique, the CNRS, and the Université Joseph Fourier, Grenoble. The costs of publication of this article were defrayed in part by the payment of page charges.…”

Section: Methodsmentioning

confidence: 99%

The Crystal Structure of the Globular Head of Complement Protein C1q Provides a Basis for Its Versatile Recognition Properties

Gaboriaud¹,

Juanhuix

Gruez³

et al. 2003

Journal of Biological Chemistry

315

366

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C1q is a versatile recognition protein that binds to an amazing variety of immune and non-immune ligands and triggers activation of the classical pathway of complement. The crystal structure of the C1q globular domain responsible for its recognition properties has now been solved and refined to 1.9 Å of resolution. The structure reveals a compact, almost spherical heterotrimeric assembly held together mainly by non-polar interactions, with a Ca 2؉ ion bound at the top. The heterotrimeric assembly of the C1q globular domain appears to be a key factor of the versatile recognition properties of this protein. Plausible three-dimensional models of the C1q globular domain in complex with two of its physiological ligands, C-reactive protein and IgG, are proposed, highlighting two of the possible recognition modes of C1q. The C1q/human IgG1 model suggests a critical role for the hinge region of IgG and for the relative orientation of its Fab domain in C1q binding.Innate immunity involves a combination of cell-surface receptors and soluble proteins with the ability to recognize microbial pathogens and thereby to generate signals that both orientate subsequent adaptive immune responses and trigger effector mechanisms (1, 2). Most of these molecules are oligomeric and recognize molecular patterns on microorganisms (3). An archetypal molecule of this type is C1q, the recognition subunit of C1, the complex that triggers activation of the classical pathway of complement, a major element of innate immunity. C1q is a 460-kDa protein with the overall shape of a bouquet of flowers, comprising six heterotrimeric collagen-like triple helices that associate in their N-terminal half to form a "stalk," then diverge to form individual "stems", each terminating in a C-terminal heterotrimeric globular domain (4). It is well documented that most of the C1 complex ligands are recognized by these peripheral globular domains, or heads, of C1q, thus triggering activation of C1r and C1s, the proteases associated with C1q (5). It is also established that C1q binds to immune complexes containing IgG or IgM, but not to those having IgA, IgD, or IgE (6). The major C1q binding site on IgG has been mapped to the CH2 domain of the Fc portion of the molecule (7-9). Although C1q shows marked differences in its reactivity toward IgG subclasses, the reason for this selectivity is not known.C1q is traditionally known for its ability to bind antibodies. However, it recognizes an amazing variety of other ligands. These include certain bacteria, viruses, parasites, and mycoplasma (6, 10 -12), underscoring its role as an antibody-independent defense protein. C1q also binds to C-reactive protein (CRP) 1 when complexed with exposed phosphocholine residues on bacteria, providing a further means of host defense (13). C1q is also capable of recognizing aberrant structures from self. Thus, in addition to cellular debris and sub-cellular membranes (14), it is established that C1q binds to, and induces clearance of, apoptotic cells (15), thereby playing a major role ...

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“…The C1q-expressing cells were expanded in the Freestyle 293 expression medium (Invitrogen) containing the selection antibiotics and 100 μg/mL L-ascorbic acid (Sigma-Aldrich), and the culture medium was harvested and replaced every 72 h up to three times. WT rC1q and rC1q variants were purified from the culture supernatant using the affinity of C1q for insoluble IgG-ovalbumin aggregates as described for isolation of C1q from human serum (22). The pH of the culture supernatant (500 mL) was adjusted to 7.0 and 8 mL of a 10 mg/mL suspension of immune aggregates was added.…”

Section: Methodsmentioning

confidence: 99%

“…C1q, proenzyme C1s-C1r-C1r-C1s, C1 inhibitor, and IgGs were purified from human serum, and their concentration was determined as described previously (21,22). Recombinant human PTX3 and MASP-3 were purified as described previously (23,24), and their molar concentration was estimated using M r values of 340,000 (25) and 87,600 (24), respectively.…”

Section: Methodsmentioning

confidence: 99%

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Expression of recombinant human complement C1q allows identification of the C1r/C1s-binding sites

Bally

Ancelet

Moriscot

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Complement C1q is a hexameric molecule assembled from 18 polypeptide chains of three different types encoded by three genes. This versatile recognition protein senses a wide variety of immune and nonimmune ligands, including pathogens and altered self components, and triggers the classical complement pathway through activation of its associated proteases C1r and C1s. We report a method for expression of recombinant full-length human C1q involving stable transfection of HEK 293-F mammalian cells and fusion of an affinity tag to the C-terminal end of the C chain. The resulting recombinant (r) C1q molecule is similar to serum C1q as judged from biochemical and structural analyses and exhibits the characteristic shape of a bunch of flowers. Analysis of its interaction properties by surface plasmon resonance shows that rC1q retains the ability of serum C1q to associate with the C1s-C1r-C1r-C1s tetramer, to recognize physiological C1q ligands such as IgG and pentraxin 3, and to trigger C1r and C1s activation. Functional analysis of rC1q variants carrying mutations of LysA59, LysB61, and/or LysC58, in the collagen-like stems, demonstrates that LysB61 and LysC58 each play a key role in the interaction with C1s-C1r-C1r-C1s, with LysA59 being involved to a lesser degree. We propose that LysB61 and LysC58 both form salt bridges with outer acidic Ca 2+ ligands of the C1r and C1s CUB (complement C1r/C1s, Uegf, bone morphogenetic protein) domains. The expression method reported here opens the way for deciphering the molecular basis of the unusual binding versatility of C1q by mapping the residues involved in the sensing of its targets and the binding of its receptors.

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Differential elution of Clq, Cl̄r and Cl̄s from human CT bound to immune aggregates. use in the rapid purification of Cl̄ sub-components

Cited by 143 publications

References 27 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

The Crystal Structure of the Globular Head of Complement Protein C1q Provides a Basis for Its Versatile Recognition Properties

Expression of recombinant human complement C1q allows identification of the C1r/C1s-binding sites

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