Autolytic Fragmentation of Complement Components C3 and C4 and Its Relationship to Covalent Binding Activity

Sim, Robert B.; Sim, Edith

doi:10.1111/j.1749-6632.1983.tb18114.x

Cited by 27 publications

(10 citation statements)

References 65 publications

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“…Another aspect of AMCOMs is the observation that heat induces exposure of the reactive thioester leading to cleavage of the protein at the thioester site (Harpel PC et al 1979;Sim and Sim 1983). In explaining the different CD109 species Lin et al proposed heat-induced degradation of the 180 kDa species to the 150 kDa species .…”

Section: Functional Aspects Of Cd109mentioning

confidence: 97%

CD109 represents a novel branch of the α2-macroglobulin/complement gene family

Solomon

Sharma

Chan

et al. 2004

Gene

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Section: Functional Aspects Of Cd109mentioning

confidence: 97%

CD109 represents a novel branch of the α2-macroglobulin/complement gene family

Solomon

Sharma

Chan

et al. 2004

Gene

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“…44,46 In addition, under experimental conditions of heat or chemical denaturation (preparing a sample for SDS-PAGE, for example), both complement and ␣2M inhibitors may undergo internal nucleophilic attack on the thioester, resulting in autolytic cleavage of the protein. [47][48][49] Although not of physiological significance, this autolytic reaction is useful diagnostically to indicate the presence of an intact thioester bond. Therefore, we determined whether native CD109 could undergo high-temperature autolytic cleavage that could be prevented by pretreatment with methylamine.…”

Section: Cd109 Is a Gpi-linked Thioester-containing Proteinmentioning

confidence: 99%

Cell surface antigen CD109 is a novel member of the α2 macroglobulin/C3, C4, C5 family of thioester-containing proteins

Lin

Sutherland

Horsfall

et al. 2002

Blood

169

163

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Cell surface antigen CD109 is a glycosylphosphatidylinositol (GPI)-linked glycoprotein of approximately 170 kd found on a subset of hematopoietic stem and progenitor cells and on activated platelets and T cells. Although it has been suggested that T-cell CD109 may play a role in antibodyinducing T-helper function and it is known that platelet CD109 carries the Gov alloantigen system, the role of CD109 in hematopoietic cells remains largely unknown. As a first step toward elucidating the function of CD109, we have isolated and characterized a human CD109 cDNA from KG1a and endothelial cells. The isolated cDNA comprises a 4335 bp open-reading frame encoding a 1445 amino acid (aa) protein of approximately 162 kd that contains a 21 aa Nterminal leader peptide, 17 potential Nlinked glycosylation sites, and a C-terminal GPI anchor cleavage-addition site. We report that CD109 is a novel member of the ␣2 macroglobulin (␣2M)/C3, C4, C5 family of thioester-containing proteins, and we demonstrate that native CD109 does indeed contain an intact thioester. Analysis of the CD109 aa sequence suggests that CD109 is likely activated by proteolytic cleavage and thereby becomes capable of thioester-mediated covalent binding to adjacent molecules or cells. In addition, the predicted chemical reactivity of the activated CD109 thioester is complementlike rather than resembling that of ␣2M proteins. Thus, not only is CD109 potentially capable of covalent binding to carbohydrate and protein targets, but the t ½ of its activated thioester is likely extremely short, indicating that CD109 action is highly restricted spatially to the site of its activation. IntroductionTo identify new surface antigens expressed by primitive hematopoietic stem and progenitor cells, we raised a series of monoclonal antibodies (mAbs) against the primitive CD34 ϩ acute myeloid leukemia cell line, KG1a. 1-3 Four of these mAbs-8A3, 7D1, 8A1, and 7C5-recognized a novel glycoprotein of approximately 170 kd that was expressed in a restricted pattern in the hematopoietic compartment and by endothelial cells. 4 Subsequently found to be identified by a number of additional mAbs, this antigen was designated CDw109 in 1993 5 and CD109 in 1996. 6 Antibodies to CD109 recognize monomeric polypeptides of about 170 kd and 150 kd in lysates of KG1a cells, T-cell lines, and activated T lymphoblasts, endothelial cells, and activated platelets. 3,[7][8][9][10][11] Peptide mapping and amino acid (aa) analysis indicate that the 150-kd form is likely derived proteolytically from the 170-kd form. 3,10 An additional band of about 120 kd that is occasionally observed arises through calcium-dependent proteolysis of the larger forms. 3,10 CD109 contains several N-linked endoglycosidase H-sensitive hybrid-type glycans but no O-linked glycans. 3,10 Consistent with this finding, ABH blood group antigens have recently been shown to be carried by platelet CD109. 12 KG1a CD109 is susceptible to cleavage with phosphatidylinositolspecific phospholipase C (PI-PLC), indicating that CD109 is bo...

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“…Some of these mutants, including C1010A in which thioester formation was formally precluded, possessed an unexpected phenotype. Although these recombinant molecules lacked haemolytic activity and the ability to form denaturationinduced autolytic fragments, as would be expected for C3 molecules lacking a thioester bond [11], they could still be cleaved by the fluid-phase C3 convertase C4b2a. Of the human thioester-bond-containing molecules, this was the first example of an ' uncoupling ' between the previously strict relationship of thioester-bond integrity and maintenance of the native conformational state.…”

Section: Introductionmentioning

confidence: 92%

Native conformations of human complement components C3 and C4 show different dependencies on thioester formation

Isaac¹,

Aivazian

Taniguchi‐Sidle

et al. 1998

Biochemical Journal

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The thioester bond in complement components C3 and C4 and the protease inhibitor alpha2-macroglobulin have traditionally been thought of as fulfilling the dual roles of mediating covalent attachment and maintaining the native conformational states of these molecules. We previously reported that several human C3 thioester-region mutants, including variants E1012Q and C1010A, in the latter of which thioester-bond formation is precluded, display an unexpected phenotype. Despite the lack of a thioester bond in these mutants, they appear to adopt a native-like conformation as suggested by the finding that they are cleavable by the classical pathway C3 convertase, C4b2a, whereas the C3b-like C3(H2O) species is not. Subsequently, a species referred to as C3(NH3)* was described which potentially could account for the observations with the above mutants. C3(NH3)* is a transient species formed on aminolysis of native C3 that can spontaneously re-form the thioester bond. Importantly, it has a mobility on cation-exchange HPLC that is distinct from both native C3 and C3(H2O), but like the native molecule, it is cleavable by an alternative-pathway C3 convertase. In this study we showed by using cation-exchange HPLC as an additional conformational probe that C3 C1010A and E1012Q mutant proteins did not resemble C3(NH3)*. Instead they displayed a chromatographic behaviour that was indistinguishable from that of native C3. To assess the general applicability of these observations, we engineered the equivalent mutations into human C4, specifically C4 C1010A and C4 E1012Q. As expected, thioester-bond formation did not occur in either of these C4 mutants, but in contrast with the results with C3 we found no evidence for the formation of a stable native-like conformation in either C4 mutant, as assessed using cleavability by C1s as the conformational probe. A possible interpretation of our data is that the adoption of the native conformational state during biosynthesis of C3 and C4 is an energetically permissible process, even if it is not locked in via thioester-bond formation. Whereas this conformational state is stable in mature C3, it is unstable in mature C4, perhaps reflecting the additional post-translational cleavage of C4 before its secretion.

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Autolytic Fragmentation of Complement Components C3 and C4 and Its Relationship to Covalent Binding Activity

Cited by 27 publications

References 65 publications

CD109 represents a novel branch of the α2-macroglobulin/complement gene family

CD109 represents a novel branch of the α2-macroglobulin/complement gene family

Cell surface antigen CD109 is a novel member of the α2 macroglobulin/C3, C4, C5 family of thioester-containing proteins

Native conformations of human complement components C3 and C4 show different dependencies on thioester formation

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