Characterization of a discontinuous epitope on annexin II by site‐directed mutagenesis

Thiel, Carsten; Weber, Klaus; Gerke, Volker

doi:10.1016/0014-5793(91)80724-h

Cited by 32 publications

(56 citation statements)

References 32 publications

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“…This configuration is similar to that observed in the structure of annexin V, which is a calcium controlled phospholipid binding protein [31]. It is noteworthy that millimolar concentations of calcium are required to saturate the binding sites in annexin II in the absence of phospholipid, but affinity for calcium is increased by more than 100-fold in its presence [32]. If the annexin model provides a valid comparator for the gelsolin system, the intramolecular calcium may play a pivotal role in regulating both actin and PIP 2 interaction.…”

Section: Discussionsupporting

confidence: 77%

Identification of the trapped calcium in the gelsolin segment 1—actincomplex: Implications for the role of calcium in the control of gelsolin activity

Weeds

Gooch

McLaughlin³

et al. 1995

FEBS Letters

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The X-ray structure of the complex of actin with gelsolin segment 1 revealed the presence of two calcium ions, one bound at an intramolecular site within segment 1 and the other bridging the segment directly to actin. Although earlier calcium binding studies at pH 8.0 revealed only a single calcium trapped in the complex (and also in the binary gelsolin-actin complex), it is here shown that two calcium ions are bound under the conditions of crystallization at physiological pH. Mutation of acidic residues in either actin or segment 1 involved in ligation of the intermolecular calcium ion resulted in loss of one of the bound calcium ions at pH < 7, but not at pH 8. Thus the calcium ion trapped in the segment 1-actin complex is that located at the intramolecular site. The implications of this for gelsolin function are discussed.

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Section: Discussionsupporting

confidence: 77%

Identification of the trapped calcium in the gelsolin segment 1—actincomplex: Implications for the role of calcium in the control of gelsolin activity

Weeds

Gooch

McLaughlin³

et al. 1995

FEBS Letters

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“…We hypothesized that, as for secretory PLA 2 s activity inhibition, cytosolic PLA 2 activity could be controlled by annexin V through a substrate depletion mechanism. Site-directed mutagenesis of annexin II and IV has been realized to study their Ca 2ϩ -binding properties to artificial vesicles; both studies demonstrate that these sites are responsible of the attachment of annexins to the vesicles (52,70). In the present study, a similar strategy of site-directed mutagenesis of the four Ca 2ϩ -binding sites of annexin V was chosen.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Cytosolic Phospholipase A2 by Annexin V in Differentiated Permeabilized HL-60 Cells

Mira

Dubois

Oudinet

et al. 1997

Journal of Biological Chemistry

107

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Annexin V belongs to a family of proteins that interact with phospholipids in a Ca 2؉ -dependent manner. This protein has been demonstrated to have anti-phospholipase A 2 activity. However, this effect has never yet been reported with the 85-kDa cytosolic PLA 2 (cPLA 2 ). We studied, in a model of differentiated and streptolysin O-permeabilized HL-60 cells, the effect of annexin V on cPLA 2 activity after stimulation by calcium, GTP␥S (guanosine 5-O-(3-thiotriphosphate)), formyl-Met-LeuPhe, or phorbol 12-myristate 13-acetate. Both recombinant and human placental purified annexin V inhibit cPLA 2 activity whatever the stimulus used. The decrease of arachidonic acid release is of 40 and 50%, respectively, at [Ca 2؉ ] of 3 and 10 M. The mechanism of inhibition was also analyzed. cPLA 2 requires calcium and protein kinase C (PKC) or mitogen-activated protein kinase phosphorylation for its activation. As annexin V was shown to be an endogenous inhibitor of PKC, PKC-stimulated cPLA 2 activity was analyzed. Using GF109203x, a specific PKC inhibitor, we demonstrated that this pathway is of minor importance in our model. cPLA 2 inhibition by annexin V is not linked to PKC inhibition. To test the hypothesis of phospholipid depletion, mutants of annexin V were constructed using mutagenesis directed to Ca 2؉ site. We demonstrate that the Ca 2؉ site located in domain I is necessary for the inhibitory effect of annexin V on cPLA 2 activity. The site in domain IV is also involved but with less efficiency. In contrast, mutations in site II and III do not modify this effect. Moreover, annexin V mutated on all sites does not inhibit cPLA 2 . Thus, we propose a predominant role of module (I/IV) in the biological action of annexin V, which, in physiological conditions, may control cPLA 2 activity by depletion of the phospholipid substrate.

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“…They are characterized by a conserved structure consisting of four repeats in annexins I-V and VII-XII, and eight repeats in annexin VI (~68 or calelectrin). Each repeat of about 70 amino acid residues contains a consensus sequence of 17 residues which is involved in Ca" binding [4,5].…”

Section: Introductionmentioning

confidence: 99%

Organisation of the chicken annexin V gene and its correlation with the tertiary structure of the protein

et al. 1993

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Chicken annexin V (anchorin CII) is a collagen binding, membrane-associated molecule with Ca" channel activity. Here we report on the coding sequences, promotor region, size and distribution of exons, and exon-intron junctions of the chicken annexin V gene. It is about 25 kb long and codes for 13 short exons between 50 and 581 bp length. Exon sizes and locations of splice sites are almost completely homologous to those of the human and mouse annexin II or pigeon annexin I genes, although there is only SO-60% homology in the sequence of the corresponding proteins. The four repeat structure and symmetry of the armexin V as evident from sequence and X-ray analysis studies is only partially reflected in this highly conserved exon dist~bution. In the first two repeats of chicken annexin V the exons correlate with protein domains ~n~ining one, two, or three a-helices, while in the repeats 3 and 4 exon junctions and o-helical domains do not correlate. The analysis of the promotor structure revealed the absence of a typical TATA-box, but a GC-rich region which may possibly promote transcription from several start sites.

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Characterization of a discontinuous epitope on annexin II by site‐directed mutagenesis

Cited by 32 publications

References 32 publications

Identification of the trapped calcium in the gelsolin segment 1—actincomplex: Implications for the role of calcium in the control of gelsolin activity

Identification of the trapped calcium in the gelsolin segment 1—actincomplex: Implications for the role of calcium in the control of gelsolin activity

Inhibition of Cytosolic Phospholipase A2 by Annexin V in Differentiated Permeabilized HL-60 Cells

Organisation of the chicken annexin V gene and its correlation with the tertiary structure of the protein

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