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Regulation of the receptor specificity and function of the chemokine RANTES (regulated on activation, normal T cell expressed and secreted) by dipeptidyl peptidase IV (CD26)-mediated cleavage.

Oravecz T, Pall M, Roderiquez G, Gorrell MD, Ditto M, Nguyen NY, Boykins R, Unsworth E, Norcross MA - J. Exp. Med. (1997)

Bottom Line: The truncated RANTES(3-68) lacked the ability of native RANTES(1-68) to increase the cytosolic calcium concentration in human monocytes, but still induced this response in macrophages activated with macrophage colony-stimulating factor.Analysis of chemokine receptor messenger RNAs and patterns of desensitization of chemokine responses showed that the differential activity of the truncated molecule results from an altered receptor specificity.Our results indicate that CD26-mediated processing together with cell activation-induced changes in receptor expression provides an integrated mechanism for differential cell recruitment and for the regulation of target cell specificity of RANTES, and possibly other chemokines.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematologic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA. tamas@helix.nih.gov

ABSTRACT
CD26 is a leukocyte activation marker that possesses dipeptidyl peptidase IV activity but whose natural substrates and immunological functions have not been clearly defined. Several chemo-kines, including RANTES (regulated on activation, normal T cell expressed and secreted), have now been shown to be substrates for recombinant soluble human CD26. The truncated RANTES(3-68) lacked the ability of native RANTES(1-68) to increase the cytosolic calcium concentration in human monocytes, but still induced this response in macrophages activated with macrophage colony-stimulating factor. Analysis of chemokine receptor messenger RNAs and patterns of desensitization of chemokine responses showed that the differential activity of the truncated molecule results from an altered receptor specificity. RANTES(3-68) showed a reduced activity, relative to that of RANTES(1-68), with cells expressing the recombinant CCR1 chemokine receptor, but retained the ability to stimulate CCR5 receptors and to inhibit the cytopathic effects of HIV-1. Our results indicate that CD26-mediated processing together with cell activation-induced changes in receptor expression provides an integrated mechanism for differential cell recruitment and for the regulation of target cell specificity of RANTES, and possibly other chemokines.

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Related in: MedlinePlus

RANTES cleavage  products after digestion with  sCD26. RANTES was incubated overnight with the indicated amounts of E+ or sCD26  and samples were subjected to  ES-MS analysis. The peaks in the  spectrum at masses (m) of 7,905  to 7,906 and 7,887 to 7,890 are  tentatively identified as [M +  K+]+ of RANTES with (7,904  daltons) and without (7,886 daltons) a molecule of H2O, respectively; the labeled peaks at the  left of the spectrum correspond  to each of these molecular ions  minus a Ser-Pro dipeptide (184  daltons).
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Figure 1: RANTES cleavage products after digestion with sCD26. RANTES was incubated overnight with the indicated amounts of E+ or sCD26 and samples were subjected to ES-MS analysis. The peaks in the spectrum at masses (m) of 7,905 to 7,906 and 7,887 to 7,890 are tentatively identified as [M + K+]+ of RANTES with (7,904 daltons) and without (7,886 daltons) a molecule of H2O, respectively; the labeled peaks at the left of the spectrum correspond to each of these molecular ions minus a Ser-Pro dipeptide (184 daltons).

Mentions: ES-MS analysis revealed that 100 nM rhRANTES underwent partial to complete hydrolysis when incubated overnight at 37°C with increasing amounts (25–250 μU) of sCD26 (Fig. 1). Taking into account cationization (K+) of the multiply charged ions, the measured molecular masses of the native and degraded polypeptides corresponded to the theoretical masses of full-length (residues 1–68) and truncated (residues 3–68) forms of RANTES, respectively. The calculated difference between the molecular masses of the native and the truncated forms ranged from 183 to 185 daltons, which is consistent with the expected mass (184 daltons) of a released Ser-Pro dipeptide, the predicted NH2 terminus of RANTES (16). In contrast to the effect of enzymatically active sCD26, shortened RANTES was not generated by incubation of the chemokine with a mutant sCD26 deficient in enzyme activity (Fig. 1). RANTES also inhibited, possibly in a competitive manner, the rapid hydrolysis of a pNA-conjugated Gly-Pro dipeptide by human placental DPPIV, as measured in a colorimetric enzyme assay (Fig. 2). The efficacy of inhibition by chemically synthesized RANTES(1–68) was similar to that observed with the DPPIV substrate and competitive inhibitor Ile-Pro-Ile (Diprotin A; reference 17), whereas RANTES(3–68) did not inhibit the reaction.


Regulation of the receptor specificity and function of the chemokine RANTES (regulated on activation, normal T cell expressed and secreted) by dipeptidyl peptidase IV (CD26)-mediated cleavage.

Oravecz T, Pall M, Roderiquez G, Gorrell MD, Ditto M, Nguyen NY, Boykins R, Unsworth E, Norcross MA - J. Exp. Med. (1997)

RANTES cleavage  products after digestion with  sCD26. RANTES was incubated overnight with the indicated amounts of E+ or sCD26  and samples were subjected to  ES-MS analysis. The peaks in the  spectrum at masses (m) of 7,905  to 7,906 and 7,887 to 7,890 are  tentatively identified as [M +  K+]+ of RANTES with (7,904  daltons) and without (7,886 daltons) a molecule of H2O, respectively; the labeled peaks at the  left of the spectrum correspond  to each of these molecular ions  minus a Ser-Pro dipeptide (184  daltons).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199148&req=5

Figure 1: RANTES cleavage products after digestion with sCD26. RANTES was incubated overnight with the indicated amounts of E+ or sCD26 and samples were subjected to ES-MS analysis. The peaks in the spectrum at masses (m) of 7,905 to 7,906 and 7,887 to 7,890 are tentatively identified as [M + K+]+ of RANTES with (7,904 daltons) and without (7,886 daltons) a molecule of H2O, respectively; the labeled peaks at the left of the spectrum correspond to each of these molecular ions minus a Ser-Pro dipeptide (184 daltons).
Mentions: ES-MS analysis revealed that 100 nM rhRANTES underwent partial to complete hydrolysis when incubated overnight at 37°C with increasing amounts (25–250 μU) of sCD26 (Fig. 1). Taking into account cationization (K+) of the multiply charged ions, the measured molecular masses of the native and degraded polypeptides corresponded to the theoretical masses of full-length (residues 1–68) and truncated (residues 3–68) forms of RANTES, respectively. The calculated difference between the molecular masses of the native and the truncated forms ranged from 183 to 185 daltons, which is consistent with the expected mass (184 daltons) of a released Ser-Pro dipeptide, the predicted NH2 terminus of RANTES (16). In contrast to the effect of enzymatically active sCD26, shortened RANTES was not generated by incubation of the chemokine with a mutant sCD26 deficient in enzyme activity (Fig. 1). RANTES also inhibited, possibly in a competitive manner, the rapid hydrolysis of a pNA-conjugated Gly-Pro dipeptide by human placental DPPIV, as measured in a colorimetric enzyme assay (Fig. 2). The efficacy of inhibition by chemically synthesized RANTES(1–68) was similar to that observed with the DPPIV substrate and competitive inhibitor Ile-Pro-Ile (Diprotin A; reference 17), whereas RANTES(3–68) did not inhibit the reaction.

Bottom Line: The truncated RANTES(3-68) lacked the ability of native RANTES(1-68) to increase the cytosolic calcium concentration in human monocytes, but still induced this response in macrophages activated with macrophage colony-stimulating factor.Analysis of chemokine receptor messenger RNAs and patterns of desensitization of chemokine responses showed that the differential activity of the truncated molecule results from an altered receptor specificity.Our results indicate that CD26-mediated processing together with cell activation-induced changes in receptor expression provides an integrated mechanism for differential cell recruitment and for the regulation of target cell specificity of RANTES, and possibly other chemokines.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematologic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892, USA. tamas@helix.nih.gov

ABSTRACT
CD26 is a leukocyte activation marker that possesses dipeptidyl peptidase IV activity but whose natural substrates and immunological functions have not been clearly defined. Several chemo-kines, including RANTES (regulated on activation, normal T cell expressed and secreted), have now been shown to be substrates for recombinant soluble human CD26. The truncated RANTES(3-68) lacked the ability of native RANTES(1-68) to increase the cytosolic calcium concentration in human monocytes, but still induced this response in macrophages activated with macrophage colony-stimulating factor. Analysis of chemokine receptor messenger RNAs and patterns of desensitization of chemokine responses showed that the differential activity of the truncated molecule results from an altered receptor specificity. RANTES(3-68) showed a reduced activity, relative to that of RANTES(1-68), with cells expressing the recombinant CCR1 chemokine receptor, but retained the ability to stimulate CCR5 receptors and to inhibit the cytopathic effects of HIV-1. Our results indicate that CD26-mediated processing together with cell activation-induced changes in receptor expression provides an integrated mechanism for differential cell recruitment and for the regulation of target cell specificity of RANTES, and possibly other chemokines.

Show MeSH
Related in: MedlinePlus