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Human macrophage-derived chemokine (MDC), a novel chemoattractant for monocytes, monocyte-derived dendritic cells, and natural killer cells.

Godiska R, Chantry D, Raport CJ, Sozzani S, Allavena P, Leviten D, Mantovani A, Gray PW - J. Exp. Med. (1997)

Bottom Line: High expression was also detected in normal thymus and less expression in lung and spleen.Unlike most other CC chemokines, MDC is encoded on human chromosome 16.MDC is thus a unique member of the CC chemokine family that may play a fundamental role in the function of dendritic cells, natural killer cells, and monocytes.

View Article: PubMed Central - PubMed

Affiliation: Icos Corporation, Bothell, Washington 98021, USA.

ABSTRACT
A cDNA encoding a novel human chemokine was isolated by random sequencing of cDNA clones from human monocyte-derived macrophages. This protein has been termed macrophage-derived chemokine (MDC) because it appears to be synthesized specifically by cells of the macrophage lineage. MDC has the four-cysteine motif and other highly conserved residues characteristic of CC chemokines, but it shares <35% identity with any of the known chemokines. Recombinant MDC was expressed in Chinese hamster ovary cells and purified by heparin-Sepharose chromatography. NH2-terminal sequencing and mass spectrophotometry were used to verify the NH2 terminus and molecular mass of recombinant MDC (8,081 dalton). In microchamber migration assays, monocyte-derived dendritic cells and IL-2-activated natural killer cells migrated to MDC in a dose-dependent manner, with a maximal chemotactic response at 1 ng/ml. Freshly isolated monocytes also migrated toward MDC, but with a peak response at 100 ng/ml MDC. Northern analyses indicated MDC is highly expressed in macrophages and in monocyte-derived dendritic cells, but not in monocytes, natural killer cells, or several cell lines of epithelial, endothelial, or fibroblast origin. High expression was also detected in normal thymus and less expression in lung and spleen. Unlike most other CC chemokines, MDC is encoded on human chromosome 16. MDC is thus a unique member of the CC chemokine family that may play a fundamental role in the function of dendritic cells, natural killer cells, and monocytes.

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Purification of recombinant MDC from CHO cells. A PCR  fragment containing bases 1–403 of the MDC cDNA was subcloned into  a mammalian expression vector driven by the CMV promoter and transfected into CHO cells. Culture supernatant from an individual transfected  clone was harvested 4 d after cells reached confluency. MDC in the media  was concentrated and purified by binding to a heparin-Sepharose column  in 0.35 M NaCl. The left panel shows the column chromatogram. Fractions collected were flowthrough (FT), 0.35 M NaCl wash (A), 0.7 M  NaCl elution (B), repeat of 0.7 M NaCl elution (C), and 1.5 M NaCl  elution (D). The right panel shows SDS-PAGE analysis (18% Tris glycine) of the supernatant loaded onto the column (L) and fractions FT, B,  C, and D. MDC eluted in fraction B (arrow). The gel was transferred to  polyvinylidene difluoride membrane, and the MDC band was excised for  NH2-terminal sequencing.
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Figure 3: Purification of recombinant MDC from CHO cells. A PCR fragment containing bases 1–403 of the MDC cDNA was subcloned into a mammalian expression vector driven by the CMV promoter and transfected into CHO cells. Culture supernatant from an individual transfected clone was harvested 4 d after cells reached confluency. MDC in the media was concentrated and purified by binding to a heparin-Sepharose column in 0.35 M NaCl. The left panel shows the column chromatogram. Fractions collected were flowthrough (FT), 0.35 M NaCl wash (A), 0.7 M NaCl elution (B), repeat of 0.7 M NaCl elution (C), and 1.5 M NaCl elution (D). The right panel shows SDS-PAGE analysis (18% Tris glycine) of the supernatant loaded onto the column (L) and fractions FT, B, C, and D. MDC eluted in fraction B (arrow). The gel was transferred to polyvinylidene difluoride membrane, and the MDC band was excised for NH2-terminal sequencing.

Mentions: MDC has 28–34% amino acid identity with other CC chemokines. It contains the characteristic four-cysteine motif in addition to nine other residues that are very highly conserved in this family (Fig. 2). Comparison with other chemokines also suggests the first 24 amino acids of MDC constitute a leader sequence, which is consistent with von Heijne's rules governing signal cleavage (45). To confirm the predicted NH2 terminus of the mature peptide, the MDC cDNA was cloned into an expression vector for stable transfection of CHO cells. Protein was purified from culture supernatants by heparin–Sepharose chromatography and gel fractionation (Fig. 3). NH2-terminal sequencing of the band migrating at the expected position of MDC yielded the sequence GPYGANMEDSV, confirming the predicted NH2 terminus of the mature protein. Amino acid analysis and mass spectrophotometry of the mature protein were consistent with the predicted amino acid composition and molecular weight (8,081 dalton) (data not shown).


Human macrophage-derived chemokine (MDC), a novel chemoattractant for monocytes, monocyte-derived dendritic cells, and natural killer cells.

Godiska R, Chantry D, Raport CJ, Sozzani S, Allavena P, Leviten D, Mantovani A, Gray PW - J. Exp. Med. (1997)

Purification of recombinant MDC from CHO cells. A PCR  fragment containing bases 1–403 of the MDC cDNA was subcloned into  a mammalian expression vector driven by the CMV promoter and transfected into CHO cells. Culture supernatant from an individual transfected  clone was harvested 4 d after cells reached confluency. MDC in the media  was concentrated and purified by binding to a heparin-Sepharose column  in 0.35 M NaCl. The left panel shows the column chromatogram. Fractions collected were flowthrough (FT), 0.35 M NaCl wash (A), 0.7 M  NaCl elution (B), repeat of 0.7 M NaCl elution (C), and 1.5 M NaCl  elution (D). The right panel shows SDS-PAGE analysis (18% Tris glycine) of the supernatant loaded onto the column (L) and fractions FT, B,  C, and D. MDC eluted in fraction B (arrow). The gel was transferred to  polyvinylidene difluoride membrane, and the MDC band was excised for  NH2-terminal sequencing.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196293&req=5

Figure 3: Purification of recombinant MDC from CHO cells. A PCR fragment containing bases 1–403 of the MDC cDNA was subcloned into a mammalian expression vector driven by the CMV promoter and transfected into CHO cells. Culture supernatant from an individual transfected clone was harvested 4 d after cells reached confluency. MDC in the media was concentrated and purified by binding to a heparin-Sepharose column in 0.35 M NaCl. The left panel shows the column chromatogram. Fractions collected were flowthrough (FT), 0.35 M NaCl wash (A), 0.7 M NaCl elution (B), repeat of 0.7 M NaCl elution (C), and 1.5 M NaCl elution (D). The right panel shows SDS-PAGE analysis (18% Tris glycine) of the supernatant loaded onto the column (L) and fractions FT, B, C, and D. MDC eluted in fraction B (arrow). The gel was transferred to polyvinylidene difluoride membrane, and the MDC band was excised for NH2-terminal sequencing.
Mentions: MDC has 28–34% amino acid identity with other CC chemokines. It contains the characteristic four-cysteine motif in addition to nine other residues that are very highly conserved in this family (Fig. 2). Comparison with other chemokines also suggests the first 24 amino acids of MDC constitute a leader sequence, which is consistent with von Heijne's rules governing signal cleavage (45). To confirm the predicted NH2 terminus of the mature peptide, the MDC cDNA was cloned into an expression vector for stable transfection of CHO cells. Protein was purified from culture supernatants by heparin–Sepharose chromatography and gel fractionation (Fig. 3). NH2-terminal sequencing of the band migrating at the expected position of MDC yielded the sequence GPYGANMEDSV, confirming the predicted NH2 terminus of the mature protein. Amino acid analysis and mass spectrophotometry of the mature protein were consistent with the predicted amino acid composition and molecular weight (8,081 dalton) (data not shown).

Bottom Line: High expression was also detected in normal thymus and less expression in lung and spleen.Unlike most other CC chemokines, MDC is encoded on human chromosome 16.MDC is thus a unique member of the CC chemokine family that may play a fundamental role in the function of dendritic cells, natural killer cells, and monocytes.

View Article: PubMed Central - PubMed

Affiliation: Icos Corporation, Bothell, Washington 98021, USA.

ABSTRACT
A cDNA encoding a novel human chemokine was isolated by random sequencing of cDNA clones from human monocyte-derived macrophages. This protein has been termed macrophage-derived chemokine (MDC) because it appears to be synthesized specifically by cells of the macrophage lineage. MDC has the four-cysteine motif and other highly conserved residues characteristic of CC chemokines, but it shares <35% identity with any of the known chemokines. Recombinant MDC was expressed in Chinese hamster ovary cells and purified by heparin-Sepharose chromatography. NH2-terminal sequencing and mass spectrophotometry were used to verify the NH2 terminus and molecular mass of recombinant MDC (8,081 dalton). In microchamber migration assays, monocyte-derived dendritic cells and IL-2-activated natural killer cells migrated to MDC in a dose-dependent manner, with a maximal chemotactic response at 1 ng/ml. Freshly isolated monocytes also migrated toward MDC, but with a peak response at 100 ng/ml MDC. Northern analyses indicated MDC is highly expressed in macrophages and in monocyte-derived dendritic cells, but not in monocytes, natural killer cells, or several cell lines of epithelial, endothelial, or fibroblast origin. High expression was also detected in normal thymus and less expression in lung and spleen. Unlike most other CC chemokines, MDC is encoded on human chromosome 16. MDC is thus a unique member of the CC chemokine family that may play a fundamental role in the function of dendritic cells, natural killer cells, and monocytes.

Show MeSH