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Polymerase chain reaction selects a novel disintegrin proteinase from CD40-activated germinal center dendritic cells.

Mueller CG, Rissoan MC, Salinas B, Ait-Yahia S, Ravel O, Bridon JM, Briere F, Lebecque S, Liu YJ - J. Exp. Med. (1997)

Bottom Line: It is induced to high levels upon spontaneous or CD40-dependent maturation and in a mixed lymphocyte reaction.In situ hybridization showed distinct expression of this gene in the germinal center.This, together with the findings that certain disintegrin metalloproteinases regulate the activity of tumor necrosis factor alpha and that metalloproteinases have also been implicated in FasL processing, suggest that this novel molecule may play an important role in dendritic cell function and their interactions with germinal center T cells.

View Article: PubMed Central - PubMed

Affiliation: Schering-Plough Laboratory for Immunological Research, 69571 Dardilly, France.

ABSTRACT
To identify genes expressed by a specific subset of dendritic cells found in vivo a polymerase chain reaction-based cDNA subtraction technique was applied to the recently described germinal center dendritic cells. A novel member of the disintegrin metalloproteinase family was cloned which comprises a not typical zinc-chelating catalytic site most similar to a bacterial metalloproteinase. Dendritic cell precursors or immature dendritic cells express no or low levels of the message. It is induced to high levels upon spontaneous or CD40-dependent maturation and in a mixed lymphocyte reaction. In situ hybridization showed distinct expression of this gene in the germinal center. This, together with the findings that certain disintegrin metalloproteinases regulate the activity of tumor necrosis factor alpha and that metalloproteinases have also been implicated in FasL processing, suggest that this novel molecule may play an important role in dendritic cell function and their interactions with germinal center T cells.

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A novel member of the disintegrin metalloproteinases. (A)  Nucleotide and amino acid sequence. The underlined nucleotides represent the original 744-bp RsaI fragment cloned from the GCDC library.  The full-length cDNA was cloned from stem cell–derived DCs. The potential polyadenylation site 17 bp upstream of the polyA tail is underlined.  Primers used in RT-PCR are shown, which generate a 562-bp product.  The first two methionine translation initiation codons are highlighted in  bold, and cysteine 187 of the conserved cysteine-switch activation mechanism is circled. The putative furin cleavage site (RISR) and the zinc-binding consensus are boxed. These sequence data are available from  EMBL/GenBank/DDBJ under the accession number Y13323. (B) Comparison of the zinc-binding sites of decysin, ScNP from Streptomyces caespitosus (20), and the five subfamilies of zinc endoproteases (38): disintegrin  metalloproteinases (snake venom H2 proteinase [39]), matrix metalloproteinases (collagenase [40]), astacin (41), serralysin (42), and thermolysin  (43). Decysin and ScNP are the only metalloproteinases in which an aspartic acid (D) replaces histidine (H) as the third zinc-chelating amino  acid. (C) Alignment of the disintegrin domains of decysin, mammalian  disintegrin metalloproteinases, and the snake disintegrin Ht-e. Decysin  displays strong amino acid conservation along the disintegrin domain before prematurely terminating. Other protein sequences continue beyond  the last cysteine. EMBL/GenBank/DDBJ accession numbers are given in  the figure.
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Figure 3: A novel member of the disintegrin metalloproteinases. (A) Nucleotide and amino acid sequence. The underlined nucleotides represent the original 744-bp RsaI fragment cloned from the GCDC library. The full-length cDNA was cloned from stem cell–derived DCs. The potential polyadenylation site 17 bp upstream of the polyA tail is underlined. Primers used in RT-PCR are shown, which generate a 562-bp product. The first two methionine translation initiation codons are highlighted in bold, and cysteine 187 of the conserved cysteine-switch activation mechanism is circled. The putative furin cleavage site (RISR) and the zinc-binding consensus are boxed. These sequence data are available from EMBL/GenBank/DDBJ under the accession number Y13323. (B) Comparison of the zinc-binding sites of decysin, ScNP from Streptomyces caespitosus (20), and the five subfamilies of zinc endoproteases (38): disintegrin metalloproteinases (snake venom H2 proteinase [39]), matrix metalloproteinases (collagenase [40]), astacin (41), serralysin (42), and thermolysin (43). Decysin and ScNP are the only metalloproteinases in which an aspartic acid (D) replaces histidine (H) as the third zinc-chelating amino acid. (C) Alignment of the disintegrin domains of decysin, mammalian disintegrin metalloproteinases, and the snake disintegrin Ht-e. Decysin displays strong amino acid conservation along the disintegrin domain before prematurely terminating. Other protein sequences continue beyond the last cysteine. EMBL/GenBank/DDBJ accession numbers are given in the figure.

Mentions: In the case of the human cell lines and stem cell-derived DCs (Fig. 2 A) 50 μg of total RNA was treated with 20 units DNAse (Boehringer Mannheim GmbH, Mannheim, Germany) in the presence of RNAse inhibitor (RNAsin; Promega, Madison, WI) in standard reaction conditions. After phenol/chloroform extraction, the RNA was reverse transcribed using Superscript™II (GIBCO BRL, Gaithersburg, MD) and a dT(12-18) oligonucleotide, according to instructions (GIBCO BRL). The reaction was stopped, and nucleic acids were ethanol precipitated and resuspended in water. About 20 ng cDNA was used in one PCR reaction. For all other cells, total RNA was prepared from ∼100,000 cells, reverse transcribed using SuperscriptTMII (GIBCO BRL) and a random hexaoligonucleotide (pd[N]6; Pharmacia) in a 20 μl reaction. 1 μl was used in a PCR reaction. The 50 μl PCR reaction contained 100 ng primers, 200 μM dNTP (Pharmacia, Uppsala, Sweden), and 0.5 units AmpliTaq™ polymerase (Roche Molecular Systems Inc., Branchburg, NJ) in standard PCR buffer (Roche Molecular Systems) and was subjected to 28 or 35 cycles of denaturing (30 s, 94°C), annealing (30 s, 60°C), and extension (2 min, 72°C) on the Perkin-Elmer thermal cycler 480. Decysin primers U137 and L677 are shown in Fig. 3 A, β actin primers were purchased (Stratagene, La Jolla, CA). The Marathon™ kit (Clontech) was used for RACE PCR and performed as recommended by the supplier. The outward primers were 5′-CCCATCAGACCAGATTTCCATACCTACC (upstream) and 5′-CCCATCTTCGGTTGCTGTTATTGAGGCT (downstream), and were used with the Marathon™ recommended cycling program 1. The two distinct PCR products were cloned into the T/A-vector and sequenced.


Polymerase chain reaction selects a novel disintegrin proteinase from CD40-activated germinal center dendritic cells.

Mueller CG, Rissoan MC, Salinas B, Ait-Yahia S, Ravel O, Bridon JM, Briere F, Lebecque S, Liu YJ - J. Exp. Med. (1997)

A novel member of the disintegrin metalloproteinases. (A)  Nucleotide and amino acid sequence. The underlined nucleotides represent the original 744-bp RsaI fragment cloned from the GCDC library.  The full-length cDNA was cloned from stem cell–derived DCs. The potential polyadenylation site 17 bp upstream of the polyA tail is underlined.  Primers used in RT-PCR are shown, which generate a 562-bp product.  The first two methionine translation initiation codons are highlighted in  bold, and cysteine 187 of the conserved cysteine-switch activation mechanism is circled. The putative furin cleavage site (RISR) and the zinc-binding consensus are boxed. These sequence data are available from  EMBL/GenBank/DDBJ under the accession number Y13323. (B) Comparison of the zinc-binding sites of decysin, ScNP from Streptomyces caespitosus (20), and the five subfamilies of zinc endoproteases (38): disintegrin  metalloproteinases (snake venom H2 proteinase [39]), matrix metalloproteinases (collagenase [40]), astacin (41), serralysin (42), and thermolysin  (43). Decysin and ScNP are the only metalloproteinases in which an aspartic acid (D) replaces histidine (H) as the third zinc-chelating amino  acid. (C) Alignment of the disintegrin domains of decysin, mammalian  disintegrin metalloproteinases, and the snake disintegrin Ht-e. Decysin  displays strong amino acid conservation along the disintegrin domain before prematurely terminating. Other protein sequences continue beyond  the last cysteine. EMBL/GenBank/DDBJ accession numbers are given in  the figure.
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Related In: Results  -  Collection

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Figure 3: A novel member of the disintegrin metalloproteinases. (A) Nucleotide and amino acid sequence. The underlined nucleotides represent the original 744-bp RsaI fragment cloned from the GCDC library. The full-length cDNA was cloned from stem cell–derived DCs. The potential polyadenylation site 17 bp upstream of the polyA tail is underlined. Primers used in RT-PCR are shown, which generate a 562-bp product. The first two methionine translation initiation codons are highlighted in bold, and cysteine 187 of the conserved cysteine-switch activation mechanism is circled. The putative furin cleavage site (RISR) and the zinc-binding consensus are boxed. These sequence data are available from EMBL/GenBank/DDBJ under the accession number Y13323. (B) Comparison of the zinc-binding sites of decysin, ScNP from Streptomyces caespitosus (20), and the five subfamilies of zinc endoproteases (38): disintegrin metalloproteinases (snake venom H2 proteinase [39]), matrix metalloproteinases (collagenase [40]), astacin (41), serralysin (42), and thermolysin (43). Decysin and ScNP are the only metalloproteinases in which an aspartic acid (D) replaces histidine (H) as the third zinc-chelating amino acid. (C) Alignment of the disintegrin domains of decysin, mammalian disintegrin metalloproteinases, and the snake disintegrin Ht-e. Decysin displays strong amino acid conservation along the disintegrin domain before prematurely terminating. Other protein sequences continue beyond the last cysteine. EMBL/GenBank/DDBJ accession numbers are given in the figure.
Mentions: In the case of the human cell lines and stem cell-derived DCs (Fig. 2 A) 50 μg of total RNA was treated with 20 units DNAse (Boehringer Mannheim GmbH, Mannheim, Germany) in the presence of RNAse inhibitor (RNAsin; Promega, Madison, WI) in standard reaction conditions. After phenol/chloroform extraction, the RNA was reverse transcribed using Superscript™II (GIBCO BRL, Gaithersburg, MD) and a dT(12-18) oligonucleotide, according to instructions (GIBCO BRL). The reaction was stopped, and nucleic acids were ethanol precipitated and resuspended in water. About 20 ng cDNA was used in one PCR reaction. For all other cells, total RNA was prepared from ∼100,000 cells, reverse transcribed using SuperscriptTMII (GIBCO BRL) and a random hexaoligonucleotide (pd[N]6; Pharmacia) in a 20 μl reaction. 1 μl was used in a PCR reaction. The 50 μl PCR reaction contained 100 ng primers, 200 μM dNTP (Pharmacia, Uppsala, Sweden), and 0.5 units AmpliTaq™ polymerase (Roche Molecular Systems Inc., Branchburg, NJ) in standard PCR buffer (Roche Molecular Systems) and was subjected to 28 or 35 cycles of denaturing (30 s, 94°C), annealing (30 s, 60°C), and extension (2 min, 72°C) on the Perkin-Elmer thermal cycler 480. Decysin primers U137 and L677 are shown in Fig. 3 A, β actin primers were purchased (Stratagene, La Jolla, CA). The Marathon™ kit (Clontech) was used for RACE PCR and performed as recommended by the supplier. The outward primers were 5′-CCCATCAGACCAGATTTCCATACCTACC (upstream) and 5′-CCCATCTTCGGTTGCTGTTATTGAGGCT (downstream), and were used with the Marathon™ recommended cycling program 1. The two distinct PCR products were cloned into the T/A-vector and sequenced.

Bottom Line: It is induced to high levels upon spontaneous or CD40-dependent maturation and in a mixed lymphocyte reaction.In situ hybridization showed distinct expression of this gene in the germinal center.This, together with the findings that certain disintegrin metalloproteinases regulate the activity of tumor necrosis factor alpha and that metalloproteinases have also been implicated in FasL processing, suggest that this novel molecule may play an important role in dendritic cell function and their interactions with germinal center T cells.

View Article: PubMed Central - PubMed

Affiliation: Schering-Plough Laboratory for Immunological Research, 69571 Dardilly, France.

ABSTRACT
To identify genes expressed by a specific subset of dendritic cells found in vivo a polymerase chain reaction-based cDNA subtraction technique was applied to the recently described germinal center dendritic cells. A novel member of the disintegrin metalloproteinase family was cloned which comprises a not typical zinc-chelating catalytic site most similar to a bacterial metalloproteinase. Dendritic cell precursors or immature dendritic cells express no or low levels of the message. It is induced to high levels upon spontaneous or CD40-dependent maturation and in a mixed lymphocyte reaction. In situ hybridization showed distinct expression of this gene in the germinal center. This, together with the findings that certain disintegrin metalloproteinases regulate the activity of tumor necrosis factor alpha and that metalloproteinases have also been implicated in FasL processing, suggest that this novel molecule may play an important role in dendritic cell function and their interactions with germinal center T cells.

Show MeSH