Shedding and gamma-secretase-mediated intramembrane proteolysis of the mucin-type molecule CD43

Abstract

CD43 is a transmembrane molecule that contains a 123-aminoacids-long cytoplasmic tail and a highly O-glycosylated extracellular domain of mucin type. Endogenous CD43 expressed in COLO 205, K562 and Jurkat cells revealed a membrane-associated, 20 kDa CD43-specific cytoplasmic tail fragment (CD43-CTF) upon inhibition of gamma-secretase. This fragment was formed by an extracellular cleavage, as it was not accumulated after treating cells with 1,10-phenanthroline, a metalloprotease inhibitor. When CD43 was transfected into HEK-293 cells expressing dominant-negative PS1 (presenilin-1), the CD43-CTF was accumulated, but not in cells with wild-type PS1. Owing to its accumulation in the presence of a non-functional PS variant, it may thus be a novel gamma-secretase substrate. This CTF is formed by an extracellular cleavage close to the membrane, is a fragment that can be concluded to be a substrate for gamma-secretase. However, the intracellular gamma-secretase product has not been possible to detect, suggesting a quick processing of this product. During normal growth the CTF was not found without gamma-secretase inhibition, but when the cells (COLO 205) were very confluent the fragment could be detected. The intracellular domain of CD43 has previously been shown to contain a functional nuclear localization signal, and has been suggested to be involved in gene activation. From this and the present results, a novel way to explain how mucin-type molecules may transduce intracellular signals can be proposed.

Figure 1. Accumulation of endogenous and expressed CD43

(A) COLO 205, Jurkat and K562 cells were incubated with the γ-secretase inhibitor (L-685,485) 18 h before lysis. The lysates were separated on a 10–20% Tris-Tricine gel, blotted and probed with αCD43-4D2 mAb followed by an alkaline phosphatase-conjugated secondary antibody and detected with the NBT/BCIP substrate. (B) CD43 (non-tagged) expression in MCF7-CD43 and SW480-CD43 cells was induced by the addition of tetracycline 24 h and incubated with the γ-secretase inhibitor (L-685,485) 18 h before lysis. The samples were analysed by Western blotting, as in (A), but instead developed by enhanced chemiluminescence after probing with an HRP-conjugated secondary antibody. The glycosylated and precursor CD43s (endogenous and induced) are indicated to the right of the gels, together with the CD43-CTF fragment generated after treatment with the γ-secretase inhibitor L-685,458. (C) Tetracycline-induced SW480-CD43 cells were treated with γ-secretase inhibitor for 18 h before lysis. Cells were also subjected to treatment with the metalloprotease inhibitors 1,10-phenanthroline and TAPI-1 for 1 and 2 h respectively. The samples were analysed as in (A) using αCD43-4D2 mAb, followed by secondary HRP-conjugated antibody and enhanced chemiluminescence detection. The membranes were re-probed with β-actin antibody followed by alkaline phosphatase-conjugated secondary antibody and detection using NBT/BCIP substrate.

Figure 2. Schematic representation of the CD43 expression plasmid constructs used and the CD43-CTF fragment generated

The epitope for the αCD43-4D2 antiserum and the location of the myc (M) and His₆ (H) tags are shown. TM, the transmembrane domain; SP, signal peptide; N247, the first CD43 amino acid in the CD43ΔE construct.

Figure 3. Generation of the CD43-CTF fragment from CD43 transfected into HEK-293 cells

(A) PS1 WT and PS1 D385N cells were transfected with pCD43MH and incubated in the presence or absence of the γ-secretase inhibitor L-685,458. Equal protein amounts of the lysates from these cells were separated on a 10–20% Tris-Tricine gel and analysed by Western blotting using αCD43-4D2 or αmyc mAb followed by an HRP-conjugated secondary mAb. (B) The same cells were transfected with pCD43 or pCD43MH 24 h before lysis. Samples were separated on a 10–20% Tris-Tricine gel followed by Western blotting using αCD43-4D2 mAb and HRP-conjugated secondary antibody and detection by enhanced chemiluminescence. The band marked with an asterisk migrating as CD43-CTF shows a degradation with loss of the myc and His₆ tags from CD43MH-CTF. (C) Membranes were prepared from PS1 WT and PS1 D385N cells transfected with pCD43MH 24 h before lysis, and incubated for 2 h at 37 °C in the presence or absence of Complete™ protease inhibitors (Roche) and 5 mM EDTA. The samples were separated on a 10–20% Tris-Tricine gel, blotted on to a PVDF membrane and probed with αCD43-4D2 mAb, followed by an alkaline phosphatase-conjugated secondary antibody and detection with NBT/BCIP substrates. (D) HEK-293 cells expressing PS1 WT or PS1 D385N were transfected with either pCD43MH or pCD43ΔE-MH. The left panel shows a Western blot using crude lysates from the cells, and the right panel shows pull-downs using Ni-NTA beads from the same lysates. The samples were separated on SDS/12.5%-PAGE gels and blotted on to a PVDF membrane, probed with the αCD43-4D2 mAb followed by an HRP-conjugated secondary mAb and detected by enhanced chemiluminescence. The precursors of the transfected proteins migrated at approx. 55 and 27 kDa respectively. The ‘CD43ct’ lane shows the migration of recombinant GST (glutathione-S-transferase)–CD43ct after purification and removal of the GST tag. CD43-CTF detected in PS1 D385N cells express full-length CD43 (CD43FL), which migrates similarly to the CD43ΔE-MH precursor. (E) HEK-293 cells expressing PS1 WT or PS1 D385N were transfected with the mutated form of either pCD43MH or pCD43ΔE-MH, as shown. Samples were separated by SDS/PAGE on 10–20% Tris-Tricine gels and blotted on to a PVDF membrane, probed with the αmyc mAb followed by an HRP-conjugated secondary mAb and detected by enhanced chemiluminescence. The CD43-CTF and CD43MH-CTF fragments and the CD43MH precursor are indicated to the right of the gels, and the degradation product lacking the myc/His₆ tags is shown by an asterisk.

Figure 4. Accumulation of CD43-CTF is dependent on cell confluence

Lysates from subconfluent and confluent COLO 205 cells, treated and not treated with the γ-secretase inhibitor L-685,458, were analysed on a 10–20% Tris-Tricine gel. The gel was blotted and probed with αCD43-4D2 mAb followed by an HRP-conjugated secondary antibody. CD43-CTF was detected regardless of γ-secretase inhibition in the confluent cells.

Figure 5. MUC1 is not cleaved by a PS1-dependent mechanism

Full-length MUC1 was transfected into HEK-293 cells expressing PS1 WT or PS1 D385N. The lysates from these cells were separated on a 10–20% Tris-Tricine gel and analysed on a Western blot using αMUC1-CT2 mAb followed by an HRP-conjugated secondary anti-hamster antibody and developed with enhanced chemiluminescence substrate. No differences in the intensity, migration or number of fragments were detected.