Immune Netw.  2011 Jun;11(3):175-181. 10.4110/in.2011.11.3.175.

Identification of CM1 as a Pathogenic Factor in Inflammatory Diseases and Cancer

• Bae S
• Kim H
• Yu YS
• Lee NE
• Kong JM
• Kim HR
• Hwang YI
• Song YW
• Kang JS
• Lee WJ

• KMID: 2150705
• DOI: http://doi.org/10.4110/in.2011.11.3.175

Abstract

BACKGROUND
CM1 (centrocyte/-blast marker 1) was defined by a mAb against concanavalin A (Con A) activated PBMC. It is expressed in germinal center of human tonsil and on the surface of activated PBMC as well as cancer cells. Recently, increased productions of pro-inflammatory mediators were detected from activated PBMC by CM1 ligation.
METHODS
However, there is a limitation to explain the exact role of CM1 on inflammation and its related mechanisms, since the identity of CM1 is still not clarified. In our previous study, we have already confirmed that soluble form of CM1 was produced by Raji. Therefore, we performed Q-TOF analysis after immunoprecipitation of concentrated Raji culture supernatant using anti-CM1 mAbs.
RESULTS
As a result, we found that CM1 is identical to enolase-1(ENO1), a glycolytic enzyme, and we confirmed that results by silencing ENO1 using siRNA. It was also confirmed through competition assay between anti-CM1 and anti-ENO1 mAbs. Finally, we investigated the possible role of CM1 in inflammatory response and cancer. The ligation of CM1 on Raji cells with anti-CM1 mAbs induces the extensive production of prostaglandin E2(PGE2). In addition, the increased activity of matrix metalloproteinase (MMP)-2/9 was shown in NCI-N87, stomach cancer cell line by CM1 stimulation.
CONCLUSION
CM1 is identical to ENO1 and it might be an important role in the regulation of inflammatory responses.

Keyword

CM1; ENO1; PGE2; Inflammatory responses; MMP-2/-9

MeSH Terms

Cell Line
Concanavalin A
Dinoprostone
Germinal Center
Humans
Immunoprecipitation
Inflammation
Ligation
Palatine Tonsil
RNA, Small Interfering
Stomach Neoplasms
Concanavalin A
Dinoprostone
RNA, Small Interfering

Figure

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  Figure 1 Immunoprecipitation of CM1 with anti-CM1 mAb. Ten liters of Raji culture supernatants were 10,000x concentrated by ultrafiltration, and then proteins, which molecular weight is more than 100 kDa or less than 30 kDa, were cuf-off by two separate steps of ultrafiltration. Hundred microliter of concentrated culture supernatants were subjected to immnoprecipitation by using twenty-five micrograms of anti-CM1 mAb and Dynabeads Protein G. Immunoprecipitated proteins were electrophoresed on 10% SDS-polyacrylamide gel and stained by silver staining kit according to manufacturer's protocol. Three bands (arrow) near at 46 kDa were selected and subject to further investigation by Q-Tof analysis.

• 

  Figure 2 Determination of CM1 as an identical molecule to ENO1. (A) Five micrograms of recombinant ENO1 protein was detected by anti-CM1 mAb. (B) Next, we investigated whether anti-CM1 mAb and anti-ENO1 mAb recognize spontaneously expressed ENO1 at the same time by the flow cytometric analysis. The binding capacity of anti-CM1 mAb (1µg) was gradually decreased by the addition of anti-ENO1 mAbs (0.1µg and 0.2µg).

• 

  Figure 3 Increase of PGE2 production by CM1 stimulation and its decreasing by ENO1 siRNA transfection. (A) Production of PGE2 was measure by ELISA as described in Materials and Methods. Briefly, cells were stimulated with anti-CM1 mAbs (1µg/ml) for 1 hr and further incubation for another 24 hrs. The amount of PGE2 produced by CM1 stimulation was measured by ELISA. (B) PGE2 production from ENO1-siRNA transfected Raji cells was measured, after stimulation with anti-CM1 mAbs.

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  Figure 4 Expression of CM1/ENO1 on NCI-N87 and its role on the induction of MMP-2/-9 production. (A) The expression of CM1/ENO1 was examined by anti-CM1 mAb and anti-ENO1 mAb, respectably. Cells were incubated with FITC-conjugated anti-CM1 mAbs or anti-ENO1 mAbs and acquired on an FACS Calibur. (B) The increase of MMP-2/-9 production by the stimulation of anti-ENO1 mAb. To examine the activity of matrix gelatinases (MMP-2 and MMP-9), NCI-N87 was incubated with anti-ENO1 mAb. Digested regions are shown as white bands on a background.

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