Functional Characterization of an Amino-terminal Region of HDAC4 That Possesses MEF2 Binding and Transcriptional Repressive Activity

Human XT-I (xylosyltransferase I; EC 2.4.2.26) initiates the biosynthesis of the glycosaminoglycan linkage region and is a diagnostic marker of an enhanced proteoglycan biosynthesis. In the present study, we have investigated mutant enzymes of human XT-I and assessed the impact of the N-terminal region on the enzymatic activity. Soluble mutant enzymes of human XT-I with deletions at the N-terminal domain were expressed in insect cells and analysed for catalytic activity. As many as 260 amino acids could be truncated at the N-terminal region of the enzyme without affecting its catalytic activity. However, truncation of 266, 272 and 273 amino acids resulted in a 70, 90 and >98% loss in catalytic activity. Interestingly, deletion of the single 12 amino acid motif G261KEAISALSRAK272 leads to a loss-of-function XT-I mutant. This is in agreement with our findings analysing the importance of the Cys residues where we have shown that C276A mutation resulted in a nearly inactive XT-I enzyme. Moreover, we investigated the location of the heparin-binding site of human XT-I using the truncated mutants. Heparin binding was observed to be slightly altered in mutants lacking 289 or 568 amino acids, but deletion of the potential heparin-binding motif P721KKVFKI727 did not lead to a loss of heparin binding capacity. The effect of heparin or UDP on the XT-I activity of all mutants was not significantly different from that of the wild-type. Our study demonstrates that over 80% of the nucleotide sequence of the XT-I-cDNA is necessary for expressing a recombinant enzyme with full catalytic activity.

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Structural and functional characterization of an epitope in the conserved C-terminal region of HIV-1 gp120

Journal of Peptide Research ◽

10.1034/j.1399-3011.1999.00082.x ◽

1999 ◽

Vol 54 (1) ◽

pp. 32-42 ◽

Cited By ~ 19

Author(s):

M. Ferrer ◽

B.J. Sullivan ◽

K.L. Godbout ◽

E. Burke ◽

H.S. Stump ◽

...

Keyword(s):

Functional Characterization ◽

Terminal Region ◽

Hiv 1

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Functional Characterization of the N-terminal Region of Myosin-2

Journal of Biological Chemistry ◽

10.1074/jbc.m605171200 ◽

2006 ◽

Vol 281 (47) ◽

pp. 36102-36109 ◽

Cited By ~ 22

Author(s):

Setsuko Fujita-Becker ◽

Georgios Tsiavaliaris ◽

Reiko Ohkura ◽

Takashi Shimada ◽

Dietmar J. Manstein ◽

...

Keyword(s):

Functional Characterization ◽

Terminal Region

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Molecular and Functional Characterization of Sex Hormone Binding Globulin in Zebrafish

Endocrinology ◽

10.1210/en.2004-0678 ◽

2004 ◽

Vol 145 (11) ◽

pp. 5221-5230 ◽

Cited By ~ 37

Author(s):

Solange Miguel-Queralt ◽

Michelle Knowlton ◽

George V. Avvakumov ◽

Rana Al-Nouno ◽

Greg M. Kelly ◽

...

Keyword(s):

Functional Characterization ◽

Intrahepatic Bile Duct ◽

Sex Hormone ◽

Sex Hormone Binding Globulin ◽

Hormone Binding ◽

Amino Terminal ◽

Stage Of Development ◽

Glycosylation Sites

Abstract SHBG (sex hormone binding globulin) transports androgens and estrogens in the blood of vertebrates including fish. Orthologs of SHBG in fish are poorly defined, and we have now obtained a zebrafish SHBG cDNA and characterized the zebrafish SHBG gene and protein through molecular biological, biochemical, and informatics approaches. Amino-terminal analysis of zebrafish SHBG indicated that its deduced precursor sequence includes a 25-residue secretion polypeptide and exhibits 22–27% homology with mammalian SHBG sequences and 41% with a deduced fugufish SHBG sequence. The 356-residue mature zebrafish SHBG (39,243 Da) sequence comprises a tandem repeat of laminin G-like domains typical of SHBG sequences; contains three N-glycosylation sites; and exists as a 105,000 ± 8700 Da homodimer. Zebrafish SHBG exhibits a high affinity and specificity for sex steroids. An RT-PCR indicated that SHBG mRNA first appears in zebrafish larva, and SHBG mRNA was localized within the liver and gut at this stage of development by whole-mount in situ hybridization. In adult fish, SHBG mRNA was found in liver, testis, and gut. In the liver, immunoreactive SHBG was present in hepatocytes and concentrated in intrahepatic bile duct cells, whereas in the testis it was confined to cells surrounding the seminiferous tubule cysts. In the intestine, immunoreactive SHBG was present in the stroma and epithelial cells of the villous projections and the surrounding muscle. The production and presence of SHBG in the gut of developing and adult zebrafish suggests a novel role for this protein in regulating sex steroid action at this site.

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Characterization of the interaction between RhoA and the amino-terminal region of PKN

FEBS Letters ◽

10.1016/0014-5793(96)00385-7 ◽

1996 ◽

Vol 385 (3) ◽

pp. 221-224 ◽

Cited By ~ 46

Author(s):

Hideki Shibata ◽

Hideyuki Mukai ◽

Yoshimasa Inagaki ◽

Yoshimi Homma ◽

Kazushi Kimura ◽

...

Keyword(s):

Terminal Region ◽

Amino Terminal

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Identification and characterization of an endogenous chemotactic ligand specific for FPRL2

Journal of Experimental Medicine ◽

10.1084/jem.20041277 ◽

2004 ◽

Vol 201 (1) ◽

pp. 83-93 ◽

Cited By ~ 73

Author(s):

Isabelle Migeotte ◽

Elena Riboldi ◽

Jean-Denis Franssen ◽

Françoise Grégoire ◽

Cécile Loison ◽

...

Keyword(s):

Defense Mechanisms ◽

Calcium Release ◽

Binding Protein ◽

Functional Characterization ◽

Formyl Peptide Receptor ◽

Specific Expression ◽

Inflammatory Reactions ◽

Amino Terminal ◽

Formyl Peptide

Chemotaxis of dendritic cells (DCs) and monocytes is a key step in the initiation of an adequate immune response. Formyl peptide receptor (FPR) and FPR-like receptor (FPRL)1, two G protein–coupled receptors belonging to the FPR family, play an essential role in host defense mechanisms against bacterial infection and in the regulation of inflammatory reactions. FPRL2, the third member of this structural family of chemoattractant receptors, is characterized by its specific expression on monocytes and DCs. Here, we present the isolation from a spleen extract and the functional characterization of F2L, a novel chemoattractant peptide acting specifically through FPRL2. F2L is an acetylated amino-terminal peptide derived from the cleavage of the human heme-binding protein, an intracellular tetrapyrolle-binding protein. The peptide binds and activates FPRL2 in the low nanomolar range, which triggers intracellular calcium release, inhibition of cAMP accumulation, and phosphorylation of extracellular signal–regulated kinase 1/2 mitogen-activated protein kinases through the Gi class of heterotrimeric G proteins. When tested on monocytes and monocyte-derived DCs, F2L promotes calcium mobilization and chemotaxis. Therefore, F2L appears as a new natural chemoattractant peptide for DCs and monocytes, and the first potent and specific agonist of FPRL2.

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