CD15 expression in human myeloid cell differentiation is regulated by sialidase activity

Abstract

The glycan determinant CD15 (also known as Lewis x, or Le(x)) is a distinguishing marker for human myeloid cells and mediates neutrophil adhesion to dendritic cells. Despite broad interest in this structure, the mechanisms underlying CD15 expression remain relatively uncharacterized. Accordingly, we investigated the molecular basis of increasing CD15 expression associated with human myeloid cell differentiation. Flow cytometric analysis of differentiating cells together with biochemical studies using inhibitors of glycan synthesis and of sialidases showed that increased CD15 expression is not due to de novo biosynthesis of CD15, but results predominantly from induction of alpha(2-3)-sialidase activity, which yields CD15 from cell-surface sialyl-CD15 (also known as sialyl-Lewis x, sLe(x) or CD15s). This differentiation-associated conversion of surface CD15s to CD15 occurs mainly on glycoproteins. Until now, modulation of post-translational glycan modifications has been attributed solely to dynamic variations in glycosyltransferase expression. Our results unveil a new paradigm by demonstrating a critical role for post-Golgi membrane glycosidase activity in the 'biosynthesis' of a key glycan determinant.

Figure 1. Hypotheses for increased CD15/Lex expression during myeloid differentiation

Structures are shown in schematic, with relevant chemical steps (arrows) for synthesis of sLe^x/CD15s and Le^x/CD15. Note that the two hypotheses are non-mutually exclusive.

Figure 2. CD44 ligation-induced changes in expression of sLe^x/CD15s and Le^x/CD15

(a) HL60 cells and (b) primary AML blasts (n=5) were treated with Hermes-1 for 72h in the presence (+) or absence (-) of 4-F-GlcNAc, and/or of DANA. Expression of Le^x/CD15 and sLe^x/CD15s was determined by flow cytometry. (b) is one representative experiment out of 5 AML specimens with analysis in triplicate cultures. Statistical significance (p≤ 0.05) for respective comparison groups is shown by brackets and asterisk.

Figure 3. CD44 ligation increases sialidase activity on myeloid cells

Cell surface sialidase activity was measured using 4-MU-NANA as substrate on (a) HL60 and KG1a cells, and (b) AML cells from patients (samples 1, 2 and 3), cultured in the presence or absence of Hermes-1 for 48h. Statistical significance (p≤ 0.05) for respective comparison groups is shown by brackets and asterisk. (c) (i): Representative ethidium bromide–stained gels of PCR-amplified RNA encoding sialidases Neu-1 and Neu-3 from HL60 cells and AML blasts treated with isotype-matched mAb (control) or Hermes-1 (48h treatment). Numbers indicate the relative expression of RT-PCR product normalized against GAPDH control. (ii): Western-blot analysis of Neu-1 protein expression in HL60 cells and AML blasts treated with isotype mAb (control) or Hermes-1 (48h treatment).

Figure 4. CD44 ligation increases Le^x/CD15 and decreases sLe^x/CD15s expression on glycoproteins of myeloid cells

(a) HL60 cells were first cultured with Hermes-1 (+) or isotype control mAb (-) for 72h, then treated with protease (bromelain) (+) or buffer alone (-) immediately before flow cytometric analysis of Le^x/CD15 and sLe^x/CD15s expression. (b) HL60 cells were treated with neuraminidase (+) and/or protease (+) or buffer (-), respectively, before flow cytometric analysis of Le^x/CD15 and sLe^x/CD15s expression. Statistical significance (p≤ 0.05) for comparison groups in (a) and (b) is shown by brackets and asterisks. (c) Western blot analysis of Le^x/CD15 and sLe^x/CD15s expression on HL60 cells treated with isotype control mAb or Hermes-1. (d) Western blot analysis of Le^x/CD15 and sLe^x/CD15s expression on PSGL-1 and CD43 immunoprecipitated from HL60 cells treated with isotype control mAb or Hermes-1. For each experiment, one representative of three is shown.

Figure 5. G-CSF treatment increases sialidase activity on myeloid cells

(a) Sialidase activity was measured from myeloid progenitor cells, before (control) and after treatment ex-vivo with G-CSF for 72h. (b) Expression of Le^x/CD15 of native progenitor cells untreated (-) or treated (+) with G-CSF for 72h and in the presence (+) or absence (-) of DANA was determined by flow cytometry using anti-CD15 mAb. One representative experiment out of three specimens with analysis in triplicate cultures is shown. (c) Typical ethidium bromide–stained gels of PCR-amplified RNA from human hematopoietic progenitor cells with or without (control) G-CSF treatment. Numbers indicate the relative expression of RT-PCR product normalized against GAPDH control.