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Purification and amino acid analysis of a human macrophage cytotoxicity-inducing factor (MCF)
C M Jones1, C A Prince, J S Williams
1Department of Medicine, University of Texas Medical School, Houston.
Abstract:
Recently we have described a CD4+ human T-cell hybridoma Ft.F3 (ATCC HB 9713). This hybridoma produces two proteins having molecular weights of 29 kd (P29) and 14.7 kd (P14.7) that function as activators of human monocyte tumor cytotoxicity and interleukin 1 (IL-1) synthesis (macrophage cytotoxicity-inducing factors, MCFs). Both MCF species were purified to apparent homogeneity, as assessed by two-dimensional (2D) gel electrophoresis, by a combination of dye ligand, ion exchange, and hydrophobic interaction chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Exhaustive treatment of P29 by endoglycosidase F, performic acid, and 40x molar excess 2-mercaptoethanol failed to generate P14.7 from P29. Antisera raised to P29 recognized only P29 in immunoblots of 2D gels of crude hybridoma supernatant. Amino acid composition analyses of both species are similar but not identical. These MCFs appear to be distinct but possibly related proteins important in the inflammatory response, whereas N-terminal analysis of P29 reveals it to be a previously undescribed cytokine.
Insights
Researchers identified two distinct proteins, P29 and P14.7, from a human T-cell hybridoma. These proteins activate monocyte tumor cytotoxicity and IL-1 synthesis, suggesting roles in inflammatory responses.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- A human T-cell hybridoma, Ft.F3, produces proteins that activate monocyte tumor cytotoxicity and IL-1 synthesis.
- These proteins are termed macrophage cytotoxicity-inducing factors (MCFs).
Purpose of the Study:
- To characterize and differentiate two MCFs, P29 and P14.7, produced by the Ft.F3 hybridoma.
- To determine if P14.7 is derived from P29 and to investigate their relationship.
Main Methods:
- Purification of P29 and P14.7 using chromatography techniques (dye ligand, ion exchange, hydrophobic interaction).
- Assessment of purity using 2D gel electrophoresis and SDS-PAGE.
- Biochemical treatments (endoglycosidase F, performic acid, 2-mercaptoethanol) to assess P29 modification.
- Immunoblotting with anti-P29 antisera.
- Amino acid composition analysis.
- N-terminal sequencing of P29.
Main Results:
- P29 and P14.7 were purified to homogeneity.
- Treatments failed to convert P29 into P14.7, indicating they are distinct.
- Anti-P29 antibodies did not cross-react with P14.7.
- Amino acid compositions were similar but not identical.
- P29 was identified as a novel cytokine.
Conclusions:
- P29 and P14.7 are distinct, albeit possibly related, proteins.
- These MCFs play a role in the inflammatory response.
- P29 represents a newly discovered cytokine involved in immune modulation.