Abstract

2-4% of all cell surface proteins are functionally regulated by a proteolytic cleavage event termed ectodomain shedding. The complement receptor 2/CD21 extracellular domain (ectodomain) is released as a soluble form constitutively as well as induced by several stimuli. Deletion of the cytoplasmic domain of CD21 augmented CD21-shedding, while removal of the extracellular membrane-adjacent short consensus repeat 16 (SCR16) abolished shedding. In the present work we show that in human cells constitutively two CD21 membrane-tethered carboxy-terminal fragments (CTFs) of 8 and 16 kDa emerge, while only the 8 kDa band was detectable in murine splenocytes. Upon proteasome inhibition the p8^CD21-CTF accumulated in the B-lymphocyte lines Raji and Daudi. In contrast, in HEK293 cells overexpressing CD21, the CTF degradation could be blocked by both lysosomal and proteasomal inhibition using chloroquine and MG132 treatment, respectively. An involvement of the lysosomal pathway was evident through accumulation of the 8 kDa band upon chloroquine treatment and co-immunoprecipitation of p8^CD21-CTF with a ubiquitin-specific antibody. However, no direct CD21 ubiquitination could be determined.

VII.2.2 Introduction

Complement receptor 2/CD21 (145 kDa) is a type I cell surface protein consisting of a large, highly glycosylated extracellular domain of 15 to 16 short consensus repeats (SCRs), a single spanning transmembrane domain and a relatively small cytoplasmic domain (262). CD21-expression is posttranscriptionally modulated by proteolytic cleavage of the extracellular domain (ectodomain), a process termed “shedding” that results in the release of soluble CD21 (sCD21) (124). sCD21 can be measured in sera of healthy donors (96,99,263) and its levels are often altered in pathologic conditions including lymphoproliferative leukemias such as B-CLL (chronic B-lymphocytic leukemia) (123,264), acute Epstein-Barr virus (EBV)-infection and other virus-associated diseases (95,124), and also in autoimmune diseases (125,126).

Although CD21 ectodomain shedding occurs constitutively, it may also be induced by B-cell receptor (BCR) crosslinking, and by the thiol antioxidants glutathione (GSH) and N-acetylcysteine (NAC) (99,107,265).

The cytoplasmic domain of CD21 consists of 34 amino acids and possesses several potential phosphorylation sites, indicating that CD21 may be a signaling molecule. The CD19/CD21/CD81 co-receptor complex enhances BCR signaling in response to complement C3d(g)-coated antigens by several orders of magnitude (21,29), and it was shown that cell signaling occurs through the long cytoplasmic tail (approximately 240 amino acids) of CD19 (52). Although the CD21 cytoplasmic region is devoid of any so far characterized functional domains, it has been implicated in CD19-independent signaling (73). The cytoplasmic domain of CD21 is involved in antigen internalization and presentation (62), B-lymphocyte signaling (73), and associates with the cytoskeleton through the formin homologue overexpressed in spleen FHOS/FHOD1 upon EBV-activation (77).

FHOS/FHOD1 is known to regulate gene transcription, actin cytoskeleton structure, and cell migration (243,244).

Several cell surface proteins regulate shedding via their cytoplasmic regions and an involvement of the actin cytoskeleton has been shown e.g. for the cell adhesion molecules L1 (245), L-selectin (246), and the hyaluronic acid receptor CD44 (148). As we have previously shown that CD21 mutants

lacking the cytoplasmic domain revealed enhanced basal and induced shedding rates, our data predict a negative regulatory role for the CD21 cytoplasmic domain, perhaps mediated through cytoskeletal anchoring to stabilize the protein in the membrane (265). In contrast, removal of the extracellular membrane adjacent SCR16 abolished basal and induced shedding rates (107).

After cleavage of the ectodomain - mostly by ADAMs (a disintegrin and metalloproteases) or MMPs (matrix metalloproteases), and in case of APP (amyloid precursor protein) alternatively by the β-secretase BACE (β-site APP cleaving enzyme) - the remaining membrane-associated carboxy-terminal fragment (CTF) is often further cleaved within the membrane by a process named regulated intramembrane proteolysis (RIP) to liberate the intracellular domain (ICD) (176). Only specific proteases are able to hydrolyze peptide bonds within the hydrophobic environment of the membrane and are therefore termed intramembrane cleaving proteases (I-CLiPs). In mammals, type I transmembrane proteins are cleaved within the membrane by the aspartyl-type γ-secretase presenilin-complex or the serine proteases rhomboids (177).

Among the I-CLiPs, only the γ-secretase complex has the exceptional ability to cleave several unrelated substrates, including APP, Notch, ErbB4, CD44, E- and N-cadherins. In contrast, rhomboids exhibit some sequence specificity towards their substrates (178). Rhomboid intramembrane cleavage is generally thought to be responsible for the release of N-terminal fragments into the extracellular space, e.g. shown for the first described rhomboid substrate, the Drosophila TGFα-like growth factor Spitz (266) and two other members of the EGF-like growth factor family, Keren and Gurken (189). The fate of the membrane-tethered CTFs after rhomboid cleavage, however, has not been investigated so far and only two mammalian rhomboid substrates are known today, EphrinB3 (193) and thrombomodulin (194).

The aim of this study was to explore whether thiol antioxidant (GSH and NAC)-induced CD21-shedding would result in the formation of CD21-CTFs and/or CD21-ICDs in CD21-transfected epithelial cells, human and murine primary lymphocytes and B-cell lines. Furthermore, the shedding-incapable mutant, CD21ΔSCR16-expressing cells were also investigated for the

VII.2.3 Results

VII.2.3.1 Two membrane-tethered C-terminal fragments (CTFs) of CD21 are constitutively present

For investigation of the C-terminal domain of CD21, a polyclonal antibody against the purified recombinant C-terminal portion of human CD21 (rCD21Cyto, 4.4 kDa) was raised, corresponding to the 34 amino acid cytoplasmic domain. With this antibody, immunoprecipitations were performed using different B-cell lines (Raji, Daudi) and HEK293 cells transfected with a CD21 wildtype construct (HEK293-CD21wt). Using Western Blot analysis, in all cell lines full length CD21 and two C-terminal fragments (CTFs) with molecular weights of approximately 8 and 16 kDa were detected (Figure 17).

Accordingly, these were named p8^CD21-CTF and p16^CD21-CTF. The two bands were also observed in isolated human peripheral blood B-lymphocytes but not in T-lymphocytes (Figure 17A, lanes 2 and 4), where only the full length protein was detectable. Interestingly, from eight healthy donors, six showed both bands (represented in lane 1, Figure 17A), while two individuals only revealed the 16 kDa band (represented in lane 3, Figure 17A). Due to the highly conserved CD21 cytoplasmic domain, the human C-terminal CD21 specific antibody could also be used to immunoprecipitate CD21-CTF from the murine CD21-transfected B-cell line CH27 and from murine splenocytes (Figure 17B). Here, the 16 kDa band was almost undetectable, even not with prolonged exposure and enhancing chemiluminescence substrate (lanes 1b and 2b).

To confirm the specificity of the immunoprecipitation, HEK293-CD21wt (data not shown) and Raji lysates were used to immunoprecipitate CD21 in a competition experiment, with or without the addition of purified rCD21Cyto (Figure 17C). Staining of both bands could be competed with rCD21Cyto, identifying p8^CD21-CTF and p16^CD21-CTF as specific C-terminal CD21 fragments.

Many sheddable cell surface proteins are further cleaved within the membrane by γ-secretase, releasing a soluble protein or peptide into the cytosol. Therefore, the question arose whether p8^CD21-CTF would be found in the cytosolic fraction. Hence, cytosolic and membrane-enriched lysates of Daudi and Raji B-cells were used for immunoprecipitation of CD21-CTF.

CD21-shedding reveals constitutively two membrane-tethered C-terminal fragments, p8^CD21-CTF and p16^CD21-CTF.

(A) CD21 was immunoprecipitated with a C-terminal specific antibody from lysates of human peripheral blood B- and T-cells from two healthy donors. Immunoblotting was performed with a second C-terminal specific CD21 antibody. (B) The murine B-cell line CH27 expressing mouse wildtype CR2/CD21 and splenocyte preparations from C57/BL6 mice were used for immunoprecipitations as described above (longer exposure times in lanes 1b and 2b). (C) CD21 was immunoprecipitated from Raji B-cells lysates as described above, or with the addition of 10 μg recombinant CD21Cyto (4.4 kDa); u. = unbound, IP = immunoprecipitation. (D) Cytosolic “C” or membrane-enriched “M” lysates of Raji and Daudi B-cells were immunoprecipitated as described above. From the same lysates, POMP was immunoprecipitated (P). (E) HEK293 cells were stably transfected with wildtype CD21 (CD21wt), or with CD21 lacking SCR16 (CD21ΔSCR16). Cells were stained with BU32 monoclonal antibody, Alexa Fluor 488-labeled secondary antibody (nuclei were stained with DAPI), and analyzed by confocal microscopy (original magnification x630). (F) Immunoprecipitations and immunoblotting was performed as described above. The basal and induced shedding properties of the CD21wt and HEK293-CD21ΔSCR16 cells are depicted below, “+” = normal, “++” = increased shedding, “-“

= reduced shedding (5-7% of wt). * indicate IgG.

In addition to full length CD21 protein, p16^CD21-CTF as well as p8^CD21-CTF were localized in the membrane fraction (Figure 17D). To control for the value of the used lysates, they were saved after the CD21-CTF immunoprecipitation and re-used for immunoprecipitation with an antibody against POMP (P), a protein known to be localized mainly to the cytoplasm and nucleus (267).

It is of note, that to detect the CD21-CTFs in one single lane in Western blot the equivalent of 450 ml blood had to be used, i.e. approximately 3x10⁷ B-cells. As a result, experiments with primary human cells were impossible and further experiments were performed with B-cell lines or HEK293-CD21wt cells.

VII.2.3.2 CD21-CTFs are constitutively present in a shedding-incapable mutant

Removal of the CD21 SCR16 has been shown to abolish CD21-shedding (107) and we were therefore interested if the CD21ΔSCR16 mutant would also produce CD21-CTFs. To verify equal expression levels, HEK293-CD21wt and HEK293-CD21ΔSCR16 cells were examined by confocal microscopy (Figure 17E), showing that both cell lines expressed comparable amounts of CD21 on the cell surface. This approach was used because the shedding-incapable mutant adhered extremely to the tissue culture flasks so that flow

cytometry analysis was an inappropriate method for determination of CD21-expression. Immunoprecipitation with the C-terminal anti-CD21 antibody revealed a shift in the molecular weight of the full length CD21wt versus CD21ΔSCR16 protein (Figure 17F). Additionally to the p8^CD21-CTF several other CTFs (approximately 10, 20, 38, 42 kDa) were constitutively present in the CD21ΔSCR16 mutant. All were membrane-associated as determined by the use of cytosolic and membrane-enriched lysates and Western blot analysis (data not shown).

VII.2.3.3 Thiol antioxidant-induced CD21-shedding does not lead to enhanced CD21-CTF levels

To study the fate of the CD21-CTFs upon shedding induction and to identify the pathway leading to their degradation, HEK293-CD21wt cells were challenged for 4 h with GSH in the presence or absence of proteasomal (MG132) or lysosomal (chloroquine) pathway inhibitors. One part of the cells was subjected to analysis of CD21 cell surface expression by flow cytometry (Figure 18A) and the other part was used for immunoprecipitation of CD21 (Figure 18A, inset). GSH alone reduced CD21 from the cell surface by more than 50%, and addition of the inhibitors enhanced CD21-shedding.

Interestingly, GSH alone did not lead to increased p8^CD21-CTF or p16^CD21-CTF levels, but in combination with MG132 and chloroquine both bands accumulated. From the cell culture supernatants, sCD21 was determined by a CD21-specific ELISA, showing increased amounts of sCD21 with GSH treatment and even more sCD21 with the inhibitors. From the remaining supernatants, sCD21 was immunoprecipitated with an antibody directed against the extracellular portion of the protein and detected with an antibody against another epitope within the approximately 130 kDa ectodomain of CD21 (98,101). In GSH- as well as in GSH + inhibitors-treated cells, the soluble form of CD21 was detectable, comparable to amounts seen with ELISA analysis (Figure 18B).

Figure 18

Thiol antioxidant-induced shedding does not lead to a significant CD21-CTF accumulation.

HEK293-CD21wt cells were stimulated for 4 h with GSH (5 mM) with or without chloroquine (Chl, 100 µM) or MG132 (50 µM). (A) Cell surface CD21-expression was determined by flow cytometry and total cell lysates immunoprecipitated as described in Figure 17 (inset) except that the upper part of the membrane was developed mAb BU32, the lower part with the C-terminal CD21 antibody. (B) Soluble CD21 (sCD21) was measured from the cell culture supernatants by a capture ELISA, and was immunoprecipitated with an antibody directed against the extracellular domain of CD21 (mAb THB5). The immunoblot was developed with mAb BU32 (inset).

Next we analyzed the concentration dependence of MG132 and chloroquine inhibitors in the presence or absence of GSH-stimulation (Figure 19A+B).

Using optimal concentrations of chloroquine (10 μM) and MG132 (50 μM), a kinetic study of the accumulation of the CD21-CTFs was performed and showed that GSH alone only led to a slight increase of p8^CD21-CTF within the 4 h incubation (Figure 19C), while GSH with MG132 strongly increased the accumulation of p8^CD21-CTF and p16^CD21-CTF (Figure 19D).

Figure 19

Kinetic of CD21-CTF accumulation upon thiol antioxidant stimulation with or without proteasomal or lysosomal pathway inhibition in HEK293-CD21wt cells.

HEK293-CD21wt cells were stimulated for 4 h with a range of Chl or MG132 concentrations (A), and additionally with GSH (5 mM) (B). HEK293-CD21wt cells were incubated for up to 4 h with 5 mM GSH alone (C), or additionally with MG132 (50 µM) (D) or chloroquine (Chl, 10 µM) (E). At the indicated time points, samples were removed for lysis and immunoprecipitation and immunoblotting with C-terminus specific CD21 antibodies (see Figure 17).

Inhibition of the lysosomal pathway by chloroquine enhanced especially accumulation of p8^CD21-CTF (Figure 19E).

To extend the data obtained in the experiment shown in Figure 18, HEK293-CD21wt cells were stimulated with various combinations of GSH and proteasomal and lysosomal inhibitors, as well as with the inhibitors alone.

Cells were subjected to analysis of CD21 cell surface expression by flow cytometry (Figure 20A) and the cell culture supernatants used for ELISA analysis (Figure 20B). The inhibitors alone or in combination had almost no effect on sCD21 levels, while together with GSH both showed significantly increased sCD21 levels. Additionally, when a combination of the inhibitors was used, a significant further elevation could be measured. GSH-mediated down-modulation of CD21 from the cell surface was only slightly enhanced with MG132, whereas chloroquine had no enhancing effect.

Proteins are often tagged by mono-ubiquitin to guide them to the lysosomal pathway (197). Since the molecular weight of approximately 16 kDa of the p16^CD21-CTF closely resembles a combination of ubiquitin (8.6 kDa) plus the 8 kDa p8^CD21-CTF, immunoprecipitations were performed with an antibody specific for mono- and/or poly-ubiquitinated proteins. HEK293-CD21wt cells were stimulated as described above with GSH, or in combination with MG132 or chloroquine. Equal amounts of the cell lysates were used for immunoprecipitation with the C-terminal specific CD21 antibody and the ubiquitin-specific antibody. Both immunoblots were developed with the second CD21 C-terminus specific antibody. Surprisingly, not the p16^CD21-CTF, but the p8^CD21-CTF was immunoprecipitated with the ubiquitin-specific antibody. The same results were obtained when the cells were stimulated with NAC (data not shown). The reverse experiment, immunoprecipitation with CD21 C-terminal specific antibody and immunoblotting with anti-ubiquitin revealed no specific bands (data not shown). However, the test with an isotype control antibody proved the specificity of this co-immunoprecipitation (Figure 20E).

This may suggest an additional ubiquitinated protein binding to the p8^CD21-CTF.

Figure 20

Thiol antioxidant-induced CD21-shedding is augmented upon proteasome inhibition in HEK293-CD21wt cells.

HEK293-CD21wt cells were stimulated with GSH (5 mM) with or without MG132 and/ or chloroquine (Chl). (A) After 4 h cells were subjected to analysis of CD21 cell surface expression by flow cytometry. The mean of 3 independent experiments is shown (+ SD; untreated control = 100%). (B) sCD21 amounts from the cell culture supernatants were determined by a capture ELISA. The mean of 8 independent experiments is shown (+ SD; untreated control = 100%). Lysate of HEK293-CD21wt cells were used for immunoprecipitation of CD21 (see Figure 17) (C), or of mono- and poly-ubiquitininated proteins using a specific mAb (D). Both blots were developed with a second CD21 C-terminal specific antibody. (E) Specificity of the co-immunoprecipitation was shown by using lysates of HEK293-CD21wt cells for immunoprecipitation with the mAb against ubiquitin, an isotype control antibody (IgG1)), or protein A sepharose alone. The membrane was probed with the C-terminal specific CD21 antibody.

VII.2.3.4 B-lymphocyte CD21-CTFs are degraded by the proteasome To analyze the above defined degradation pathways for the CD21-CTFs in B-lymphocytes, the same set of experiments was performed with Daudi and Raji B-cells. Stimulation of Daudi B-cells showed an approximately 30% reduction of cell surface CD21 within 4 h of stimulation with NAC (to 71.5 ± 4.6%) or GSH (to 70.5 ± 4.0%) (Figure 21A), and increased amounts of soluble CD21

could be immunoprecipitated from the cell culture supernatant (Figure 21B).

Soluble CD21 was detectable even without shedding induction, showing that CD21-shedding is a spontaneous event in these cells. Stimulation of the cells with NAC or GSH however, led to augmented sCD21 levels. Total lysate of Daudi cells was loaded as well as the immunoprecipitated soluble CD21, demonstrating the presence of the two isoforms of CD21, sCD21 and mCD21 (membrane CD21).

In both Raji and Daudi cells, the lysosomal pathway inhibitor chloroquine did not have an effect on p8^CD21-CTF (Figure 21C+D), and immunoprecipitation with the ubiquitin-specific antibody did not reveal co-immunoprecipitated CD21, even not following prolonged exposure (data not shown). Proteasomal pathway inhibition on the other hand, revealed a concentration-dependent sensitivity of the cells with regard to p8^CD21-CTF accumulation. The effect was independent of GSH-induced shedding, where neither p8^CD21-CTF nor p16

CD21-CTF accumulated. In conclusion, in B-cells, the 8 kDa CD21-CTF does not accumulate upon GSH-induced shedding or inhibition of the lysosomal pathway, but on proteasome inhibition.

Figure 21

CD21-CTFs do not accumulate upon thiol antioxidant-induced CD21-shedding, but upon proteasome inhibition in B-cells.

Daudi B-cells were challenged with NAC or GSH (both 5 mM). (A) After 4 h cells were subjected to analysis of CD21 cell surface expression by flow cytometry. The mean of 4 independent experiments is shown (+ SD; untreated control = 100%). (B) From the cell culture supernatants, sCD21 was immunoprecipitated with mAb THB5 and the Western blot developed with mAb BU32. In lane 5, Daudi cell lysate was loaded, to compare the molecular weight of membrane CD21 (mCD21) and soluble CD21 (sCD21). Daudi and Raji B-cell lines were treated with GSH (5 mM) alone or with combinations of different MG132 or chloroquine (Chl) concentrations. After 4 h cells were lysed and immunoprecipitated as described in Figure 17. (C) Raji, and (D) Daudi B-cells. After immunoprecipitation of CD21, the lysates were re-used for immunoprecipitation of a housekeeping protein (POMP, P).

VII.2.4 Discussion

In the eukaryotic cell, two major systems for the regulation of protein stability and degradation exist. The standard pathway for degradation of intracellular proteins is the proteasome, and for extracellular and transmembrane proteins, the lysosomes (221). The transmembrane protein CD21 was previously shown to be internalized upon crosslinking with the B-cell antigen receptor (BCR) (62) and gold-labeled C3dg has been traced into different compartments of the endocytic pathway, co-localizing with HLA-DR MHC class II molecules (92). Here, we define that CD21-CTFs are degraded by the proteasomal pathway in B-lymphocytes. When overexpressed in epithelial cells, both lysosomal and proteasomal degradation pathways may have been used by the cells to dispose of excess protein. We found that CD21-CTFs were present in human as well as in murine B-lymphocytes.

Two membrane-tethered CD21-CTFs, p8^CD21-CTF and p16^CD21-CTF, were constitutively present in human B-cells, as well as in HEK293 cells overexpressing the protein. It is noteworthy, that in two out of eight human subjects analyzed the 16 kDa CTF was missing, suggesting dynamic differences. In T-cells, suspected not to contribute to the sCD21-pool found in plasma (99), neither band was detectable, while the full length protein was seen. The relatively low CD21-expression levels in these cells may be explained by the fact that the highest CD21-expression measured on T-cells was reported to be on immature, double negative (CD4^-/CD8^-) thymocytes (20).

Using a ubiquitin-specific antibody, we were able to co-precipitate p8^CD21-CTF in the CD21-overexpressing HEK293 cells, but not p16^CD21-CTF. Since ubiquitin is a 8.6 kDa peptide this suggests that p8^CD21-CTF might be associated with other ubiquitinated proteins. In mice, however, only the smaller 8 kDa CTF was detectable.

A CD21-peptide of 8 kDa could be derived from the cytosolic and transmembrane (TM) domains of the protein. This would be in agreement with previous results suggesting shedding of CD21 to occur close to the

A CD21-peptide of 8 kDa could be derived from the cytosolic and transmembrane (TM) domains of the protein. This would be in agreement with previous results suggesting shedding of CD21 to occur close to the