Investigation of active form of catalytic antibody light chain 41S-2-L

Differentiation of stromal cells into decidual cells, which is critical to successful pregnancy, represents a complex transformation requiring changes in cytoskeletal architecture. We demonstrate that in vitro differentiation of human uterine fibroblasts into decidual cells includes down-regulation of α-smooth muscle actin and β-tubulin, phosphorylation of focal adhesion kinase, and redistribution of vinculin. This is accompanied by varied adhesion to fibronectin and a modified ability to migrate. Cytoskeletal organization is determined primarily by actin-myosin II interactions governed by the phosphorylation of myosin light chain (MLC20). Decidualization induced by cAMP [with estradiol-17β (E) and medroxyprogesterone acetate (P)] results in a 40% decrease in MLC20 phosphorylation and a 55% decline in the long (214 kDa) form of myosin light-chain kinase (MLCK). Destabilization of the cytoskeleton by inhibitors of MLCK (ML-7) or myosin II ATPase (blebbistatin) accelerates decidualization induced by cAMP (with E and P) but inhibits decidualization induced by IL-1β (with E and P). Adenoviral infection of human uterine fibroblast cells with a constitutively active form of MLCK followed by decidualization stimuli leads to a 30% increase in MLC20 phosphorylation and prevents decidualization. These data provide evidence that the regulation of cytoskeletal dynamics by MLC20 phosphorylation is critical for decidualization.

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Regulation of cytoskeletal mechanics and cell growth by myosin light chain phosphorylation

AJP Cell Physiology ◽

10.1152/ajpcell.1998.275.5.c1349 ◽

1998 ◽

Vol 275 (5) ◽

pp. C1349-C1356 ◽

Cited By ~ 75

Author(s):

Shuang Cai ◽

Lidija Pestic-Dragovich ◽

Martha E. O’Donnell ◽

Ning Wang ◽

Donald Ingber ◽

...

Keyword(s):

Cell Growth ◽

Map Kinase ◽

Light Chain ◽

Intracellular Signaling ◽

Myosin Light Chain ◽

Active Form ◽

Fold Increase ◽

Mitogen Activated Protein Kinase ◽

Apical Surface ◽

Myosin Light Chain Phosphorylation

The role of myosin light chain phosphorylation in regulating the mechanical properties of the cytoskeleton was studied in NIH/3T3 fibroblasts expressing a truncated, constitutively active form of smooth muscle myosin light chain kinase (tMK). Cytoskeletal stiffness determined by quantifying the force required to indent the apical surface of adherent cells showed that stiffness was increased twofold in tMK cells compared with control cells expressing the empty plasmid (Neo cells). Cytoskeletal stiffness quantified using magnetic twisting cytometry showed an ∼1.5-fold increase in stiffness in tMK cells compared with Neo cells. Electronic volume measurements on cells in suspension revealed that tMK cells had a smaller volume and are more resistant to osmotic swelling than Neo cells. tMK cells also have smaller nuclei, and activation of mitogen-activated protein kinase (MAP kinase) and translocation of MAP kinase to the nucleus are slower in tMK cells than in control cells. In tMK cells, there is also less bromodeoxyuridine incorporation, and the doubling time is increased. These data demonstrate that increased myosin light chain phosphorylation correlates with increased cytoskeletal stiffness and suggest that changing the mechanical characteristics of the cytoskeleton alters the intracellular signaling pathways that regulate cell growth and division.

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In VitroSelection of Fab Fragments by mRNA Display and Gene-Linking Emulsion PCR

Journal of Nucleic Acids ◽

10.1155/2012/371379 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 19

Author(s):

Takeshi Sumida ◽

Hiroshi Yanagawa ◽

Nobuhide Doi

Keyword(s):

Light Chain ◽

Active Form ◽

Translation System ◽

Mrna Display ◽

Single Chain ◽

Fab Fragment ◽

Fab Fragments ◽

Emulsion Pcr ◽

Overlap Extension

In vitroselection by display methods has been an effective tool for engineering recombinant antibodies. mRNA display based on a cell-free translation system has the advantages of larger library sizes and quicker selection procedures compared with cell-based display methods such as phage display. However, mRNA display has been limited to select single-chain polypeptides such as scFvs due to its characteristic of linking a nascent polypeptide with its encoding mRNA on the ribosome. Here we demonstrated a new way of selecting heterodimeric Fab fragments by using mRNA display combined with emulsion PCR. We designed a pair of complementary 5′ UTR sequences that can link the Fab heavy and light chain genes together by overlap-extension PCR in water-in-oil emulsions. We confirmed that two mRNA-displayed polypeptides for heavy and light chain of a model Fab fragment were associated into the active form and that a specific Fab fragment gene was enriched over 100-fold per round of a model affinity selection followed by the gene-linking emulsion PCR. We further performed directed evolution of Fab fragments with higher binding activity from a randomized Fab fragment library.

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Localization of Factor VIII Interactive Site within Plasmin/Plasminogen Which Is Responsible for Plasmin-Catalyzed Activation/Inactivation of Factor VIII.

Blood ◽

10.1182/blood.v110.11.1759.1759 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1759-1759 ◽

Cited By ~ 1

Author(s):

Katsumi Nishiya ◽

Keiji Nogami ◽

Kiyotaka Okada ◽

Osamu Matsuo ◽

Masahiro Takeyama ◽

...

Keyword(s):

Factor Viii ◽

Light Chain ◽

Catalytic Domain ◽

Active Form ◽

Coagulation Factors ◽

Competitive Binding Assay ◽

Key Enzyme ◽

Proteolytic Cleavages ◽

20 Nm ◽

A2 Domain

Abstract Plasmin (Plm), an active form of plasminogen (Plg), not only functions as a key enzyme in the fibrinolytic system, but also directly inactivates several coagulation factors. Especially, factor VIII is inactivated by Plm immediately after the activation by limited proteolytic cleavage at Lys36, Arg336, Arg372, and Arg740 in the heavy chain, and at Arg1689 and Arg1721 in the light chain (Nogami et al. J. Biol. Chem. 2007, 282, 5287). We recently have identified the plasmin-interactive sites on the A2 domain responsible for cleavages at Arg336 and Arg372, and on the light chain responsible for cleavage at Lys36 (Abst #1991/1709, BLOOD 102/108, 2005/2006). In the present study, we attempted to localize a factor VIII-interactive site on Plm (and Plg). Competitive binding assay using 6-aminohexanoic acid (6-AHA), a competitor of lysine-binding site (LBS) of Plm/Plg, showed that 6-AHA markedly inhibited (by >90%) the light chain binding to active-site modified Plm (anhydro-Plm), whilst inhibited weakly the A2 binding (by ∼30%). These results suggested that the light chain interaction with Plm was mainly dependent upon LBS, but the A2 interaction was independent. The addition of monoclonal antibody (mAb) against Plg kringle 5-catalytic domain (K5-CD) significantly inhibited Plm-catalyzed activation/inactivation of factor VIII or VIIIa with an ∼4-fold lower rate constant. On the other hand, anti-K1-3 and anti-K4 mAbs any little affected. SDS-PAGE analysis revealed that only anti-K5-CD mAb blocked Plm-catalyzed cleavages at Arg336 and Arg372 by ∼90% in dose-dependent manners (IC50: ∼20 nM). Surface plasmon resonance-based assays showed that the isolated K5-CD bound to factor VIII with an ∼50-fold higher affinity (Kd: 3 nM) compared to the K1-3 and K4, similar to the affinity obtained with anhydro-Plm (Kd: 4 nM). In particular, the K5-CD bound to the A2 domain with an ∼5-fold higher affinity (Kd: 42 nM) than those obtained with the K1-3 and K4. In contrast, both the K1-3 and K4 bound to the light chain predominantly (Kd: 43 and 87 nM, respectively), whilst the K5-CD failed to bind. Furthermore, the addition of a goat antibody against the CD (C-14; Santa Cruz Biotechnology) completely blocked the A2 and K5-CD interaction (by ∼95%). These findings suggest that the CD of Plm (and Plg) interacts with the factor VIII A2 domain through the LBS-independent mechanisms, whilst the K1-3 (and/or K4) interacts with the light chain through the LBS-dependent mechanisms. Furthermore, the CD and A2 interaction would regulate the activation/inactivation of factor VIII by proteolytic cleavages of Arg336 and Arg372.

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New technologies to introduce a catalytic function into antibodies: A unique human catalytic antibody light chain showing degradation of β-amyloid molecule along with the peptidase activity

FASEB BioAdvances ◽

10.1096/fba.1025 ◽

2019 ◽

Vol 1 (2) ◽

pp. 93-104

Author(s):

Emi Hifumi ◽

Hiroaki Taguchi ◽

Eiichi Toorisaka ◽

Taizo Uda

Keyword(s):

Light Chain ◽

New Technologies ◽

Catalytic Antibody ◽

Peptidase Activity ◽

Catalytic Function ◽

Β Amyloid

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An increase or a decrease in myosin II phosphorylation inhibits macrophage motility.

The Journal of Cell Biology ◽

10.1083/jcb.114.2.277 ◽

1991 ◽

Vol 114 (2) ◽

pp. 277-283 ◽

Cited By ~ 54

Author(s):

A K Wilson ◽

G Gorgas ◽

W D Claypool ◽

P de Lanerolle

Keyword(s):

Cell Motility ◽

Light Chain ◽

Mammalian Cells ◽

Inverse Relationship ◽

Myosin Light Chain ◽

Myosin Ii ◽

Active Form ◽

Chemotaxis Assay ◽

Mlc20 Phosphorylation ◽

Number Of Cells

Myosin II purified from mammalian non-muscle cells is phosphorylated on the 20-kD light chain subunit (MLC20) by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK). The importance of MLC20 phosphorylation in regulating cell motility was investigated by introducing either antibodies to MLCK (MK-Ab) or a Ca2+/calmodulin-independent, constitutively active form of MLCK (MK-) into macrophages. The effects of these proteins on cell motility were then determined using a quantitative chemotaxis assay. Chemotaxis is significantly diminished in macrophages containing MK-Ab compared to macrophages containing control antibodies. Moreover, there is an inverse relationship between the number of cells that migrate and the amount of MK-Ab introduced into cells. Interestingly, there is also an inverse relationship between the number of cells that migrate and the amount of MK- introduced into cells. Other experiments demonstrated that MK-Ab decreased intracellular MLC20 phosphorylation while MK- increased MLC20 phosphorylation. MK- also increased the amount of myosin associated with the cytoskeleton. These data demonstrate that the regulation of MLCK is an important aspect of cell motility and suggest that MLC20 phosphorylation must be maintained within narrow limits during translational motility by mammalian cells.

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Lysine-Binding Site-Dependent Interaction with Plasmin and Factor Va Responsible for Plasmin-Catalyzed Factor Va Inactivation.

Blood ◽

10.1182/blood.v112.11.2016.2016 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2016-2016

Author(s):

Katsumi Nishiya ◽

Keiji Nogami ◽

Kiyotaka Okada ◽

Osamu Matsuo ◽

Kenichi Ogiwara ◽

...

Keyword(s):

Binding Site ◽

Heavy Chain ◽

Light Chain ◽

Active Form ◽

Limited Proteolysis ◽

Coagulation Factor ◽

Dependent Manner ◽

Competitive Binding Assay ◽

Factor Va ◽

Sds Page

Abstract Plasmin (Plm), an active form of plasminogen (Plg), functions as a key enzyme in the fibrinolytic system. Furthermore, this enzyme directly inactivates various coagulation factors such as factor V (FV) and factor VIII (FVIII) by limited proteolysis, suggesting another role of Plm in the regulation of the coagulation system. We recently reported that Plm/Plg interacts with FVIII and its active form (FVIIIa), both dependently and independently of lysine-binding site (LBS) (Blood2007; 110, 522a). In this study, we attempted to localize a factor Va (FVa)-interactive region on Plm (and Plg) using Plm/Plg kringle fragments. Surface plasmon resonance-based assays showed that FVa directly bound to active-site modified Plm (anhydro-Plm) with an ~2-fold higher affinity, compared to Plg (Kd; 97 and 198 nM, respectively). In particular, FVa bound to the immobilized-Plg fragment consisting of kringle 1-2-3 domains (K1-3) (Kd: 706 nM), whilst FVa failed to bind both the kringle 4 domain (K4) and Plg fragment consisting of kringle 5 and catalytic domains (K5-CD). A similar experiment using immobilized FVa also revealed that the K1-3 solely bound to FVa. These results were quite different from those obtained by FVIII and Plm/Plg binding experiment that the K5-CD bound to FVIII(a) more preferably. Competitive binding assay using 6-aminohexanoic acid (6-AHA), a competitor of LBS of Plm/Plg, showed that 6-AHA markedly inhibited (by >90%) the K1-3 binding to FVa (IC50; ~25 μM), suggesting that interaction of FVa with Plm is mostly dependent upon LBS. According to the one stage-clotting assay, 6-AHA inhibited (>90%) Plm-catalyzed inactivation of FVa in a dose-dependent manner (IC50; ~10 μM). Furthermore, Plm-catalyzed inactivation of FVa was blocked by an anti-K1-3 monoclonal antibody (mAb), not by either anti-K4 or anti-K5-CD mAb, although Plm-catalyzed inactivation of FVIII was blocked by anti-K5-CD mAb. In order to confirm that the inhibitory effect of 6-AHA on the Plm-catalyzed inactivation, we performed SDS-PAGE experiment. Plm cleaves FVa at Lys309 and Arg348 in the heavy chain, and at Arg1752 in the light chain. SDS-PAGE analysis revealed that 6-AHA blocked Plm-catalyzed cleavages of the light chain more prominently than that of the heavy chain (IC50; ~10 and ~>100 μM, respectively). These findings suggest that the K1-3 of Plm (and Plg) interacts with the FVa through the LBS-dependent mechanisms, and these interactions likely contribute to the FVa-catalyzed inactivation by proteolytic cleavages at Arg1752 in the light chain. Present study indicated that plasmin-catalyzed protelytic inactivation of coagulation factor is complicatedly regulated by the LBS dependency in the protein and protein interaction.

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