Functional analysis of the cytoplasmic domain of complement receptor type II (CR2/CD21)

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Functional analysis of the

cytoplasmic domain of complement receptor type II (CR2/CD21)

Dissertation

zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr.rer.nat)

an der Universität Konstanz (Fachbereich Biologie)

Vorgelegt von

Melanie Merja Hoefer

Tag der mündlichen Prüfung: 22. März 2006

1. Referent: Prof. Dr. Daniel Dietrich 2. Referent: Prof. Dr. Harald Illges

Konstanz, 2006

Konstanzer Online-Publikations-System (KOPS) URL: http://www.ub.uni-konstanz.de/kops/volltexte/2007/1799/

URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-17998

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If we knew what it was We were doing,

It would not be called Research, Would it?

Albert Einstein

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To my family

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I T ABLE OF CONTENTS

I TABLE OF CONTENTS ...1

II ABBREVIATIONS ...3

III SUMMARY ...6

IV DEUTSCHE ZUSAMMENFASSUNG...8

V INTRODUCTION ...10

V.1 Complement receptor type II (CR2/CD21) ...10

V.1.1 CD21 in innate and adaptive immunity...11

V.1.2 The role of the cytoplasmic domain of CD21 ...13

V.1.3 Primary and secondary structure of the cytoplasmic domain of CD21, posttranslational modifications ...13

V.1.4 Interacting partners of the cytoplasmic domain of CD21...16

V.1.5 CD21 and antigen presentation ...18

V.1.6 Soluble CD21...20

V.1.7 Regulation of CD21-shedding ...21

V.1.8 Functional consequences of CD21-shedding...23

V.1.9 CD21 and sCD21 in disease...24

V.2 Ectodomain shedding and involved proteases ...26

V.2.1 Matrix metalloproteases and ADAMs ...26

V.2.2 BACE, the beta-site amyloid precursor protein secretase...27

V.2.3 Serine proteases involved in shedding...28

V.3 Intramembrane cleaving proteases (I-CLiPs) and regulated intramembrane proteolysis (RIP) ...30

V.3.1 The γ-secretase complex ...32

V.3.2 The rhomboid family of serine proteases ...35

V.4 Lysosomal degradation and endocytosis of membrane proteins ...36

V.5 Proteasomal degradation of proteins and the role of POMP in proteasome maturation...39

V.5.1 Proteasome function ...39

V.5.2 The proteasome maturation factor POMP...42

V.5.3 Proteasome localization ...43

VI ADDRESSED QUESTIONS AND AIMS OF THIS STUDY ...45

VII RESULTS ...46

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VII.1 Augmented ectodomain shedding of the murine complement receptor type 2 (CR2/CD21) in the absence of its cytoplasmic

domain...46

VII.1.1 Summary...47

VII.1.2 Introduction ...48

VII.1.3 Results ...50

VII.1.4 Discussion...61

VII.1.5 Material and methods...65

VII.2 Constitutive generation of two carboxy-terminal fragments (CTFs) of CD21...68

VII.2.1 Abstract...69

VII.2.5 Material and methods...85

VII.3 Possible tetramerization of the proteasome maturation factor POMP/proteassemblin/hUmp1 and its sub-cellular localization ...90

VII.3.1 Abstract...91

VII.3.3 Materials and methods...94

VIII DISCUSSION ...117

VIII.1 The role of the cytoplasmic domain in CD21-shedding ...117

VIII.1.1 The influence of the cytoplasmic domain on CD21-shedding....117

VIII.1.2 Pervanadate modulates CD21-expression on mouse B-cells....119

VIII.1.3 CD21-sheddases...119

VIII.1.4 Two CD21-CTFs are constitutively present and degraded by the proteasomal pathway...121

VIII.1.5 Does CD21 undergo RIP?...123

VIII.1.6 CD21-shedding and T-cells ...125

VIII.2 The proteasome maturation factor POMP – a challenging protein 127 VIII.2.1 POMP features interesting physicochemical properties ...127

VIII.2.2 POMP may act as tetramer ...128

VIII.2.3 POMP may reside within PML-NBs in malignant cells...129

VIII.2.4 POMP-expression in malignant and non-malignant cells ...129

IX REFERENCES ...131

X PUBLICATIONS...151

XI RECORD OF ACHIEVEMENT/ EIGENABGRENZUNG...152

XII ACKNOWLEDGEMENTS ...153

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II A BBREVIATIONS

aa amino acid AAT α1-antitrypsin

Ab antibody

AD Alzheimer’s disease

ADAM a disintegrin and metalloprotease

Ag antigen

APP amyloid precursor protein ATL adult T-cell leukemia ATP adenosine triphosphate BAFF B-cell activation factor Bcl-2 B-cell leukemia/lymphoma-2

B-CLL B-cell-type chronic lymphocytic leukemia BCR B-cell antigen receptor

BLys B-lymphocyte stimulator bp base pairs

C3d, C3dg complement (C) 3 fragments CD cluster of differentiation

Chl chloroquine diphosphate (C18H26ClN3·2H3PO4) CLL chronic lymphocytic leukemia

CML chronic myeloid leukemia CR complement receptor CTF carboxy-terminal fragment EBV Epstein-Barr virus

ECM extracellular matrix ER endoplasmic reticulum FDC follicular dendritic cell

FHOS formin homologue overexpressed in spleen GC germinal center

GSK3 glycogen synthase kinase-3 GSH reduced glutathione

GSSG oxidized glutathione; glutathione disulfide GST glutathione S-transferase

HEL hen-egg lysozyme HLA human leukocyte antigen hUmp1 human Ump1 homologue iC3b complement (C) 3 fragment ICD intracellular domain

I-CLiP intramembrane cleaving protease IFN interferon

Ig immunoglobulin IL interleukin

IPTG isopropyl β-D-1-thiogalactopyranoside kDa kilo Dalton

mAb monoclonal antibody MDa mega Dalton

MG132 N-[(Phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3

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MHC major histocompatibility complex MMP matrix metalloprotease

MMPi MMP inhibitor

MT-MMP membrane-type MMP MW molecular weight NAC N-acetylcysteine NF-κB nuclear factor κB NK natural killer cells

PA28 28 kDa proteasome activator PBMC peripheral blood mononuclear cells PMA phorbol-12-myristate-13-acetate POMP proteasome maturation protein pSS primary Sjögren`s syndrome

PV pervanadate

RA rheumatoid arthritis RING really interesting new gene

RIP regulated intramembrane proteolysis ROS reactive oxygen species

SCR short consensus repeat SH Src homology

SLE systemic lupus erythematosus Sp100 100 kDa speckled protein SUMO small ubiquitin like modifier TACE TNF-α converting enzyme

TAP transporter associated with antigen processing TCR T-cell antigen receptor

TGF-α transforming growth factor-α

TIMP tissue inhibitor of metalloproteinases TMD transmembrane domain

TNF-α tumor necrosis factor-α UIM ubiquitin interacting motif Ump1(p) yeast POMP homologue wt wildtype

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Amino acids

Alanine Ala A

Arginine Arg R

Asparagine Asn N

Aspartic acid Asp D

Cysteine Cys C

Glutamine Gln Q

Glutamic acid Glu E

Glycine Gly G

Histidine His H

Isoleucine Ile I

Leucine Leu L

Lysine Lys K

Methionine Met M

Phenylalanine Phe F

Proline Pro P

Serine Ser S

Threonine Thr T

Tryptophan Trp W

Tyrosine Tyr Y

Valine Val V

any aa X

hydrophobic aa h

aa with bulky hydrophobic group Ø

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III S ^UMMARY

The complement receptor type 2 (CR2/CD21) is functionally regulated by a proteolytic cleavage event termed “ectodomain shedding” which results in the release of the extracellular domain as functional active, soluble protein. Deletion of the extracellular membrane-adjacent short consensus repeat 16 (SCR16) abolished shedding.

The aim of this work was to investigate the role of the cytoplasmic domain in CD21-shedding. Shedding was induced either by the thiol antioxidants glutathione and N-acetylcysteine or by activation of the cells through the phosphotyrosine-specific phosphatase inhibitor pervanadate. A negative regulatory role for the CD21 cytoplasmic domain could be established, as murine B-cells expressing CD21 mutants lacking this domain revealed higher basal and induced shedding rates. Interestingly, CD21 cell surface expression was up-regulated upon stimulation with low (20 μM) pervanadate concentrations, while higher (200 μM) concentrations lead to CD21-shedding.

This prompted us to suggest CD21-shedding as mechanism to fine-tune B-cell activation.

It was of further interest, whether CD21 would undergo regulated intramembrane proteolysis (RIP), a process often taking place after initial ectodomain shedding. In fact, in human B-lymphocytes and several B-cell lines, two constitutively present, membrane-tethered carboxy-terminal fragments were identified, p8^CD21-CTF and p16^CD21-CTF. Human T-lymphocytes, suspected not to shed CD21, featured neither CTF, although the full length protein could be detected. In murine splenocytes, only the smaller p8^CD21-CTF was seen. In the SCR16-lacking mutant the p8^CD21-CTF was still present, suggesting that CD21 could be cleaved within the membrane, independent of ectodomain shedding. In B-cells, the p8^CD21-CTF was degraded by the proteasomal pathway, independent of additional shedding induction by thiol antioxidants. Moreover, when CD21 was overexpressed in HEK293 epithelial cells, the two CD21-CTFs were degraded both by the proteasomal and the lysosomal pathways.

To identify potential intracellular binding partners, the CD21 cytoplasmic domain was used in a Yeast Two-Hybrid screening. One candidate, the proteasome

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maturation protein POMP, was further investigated. Although the interaction with CD21 only occurred in yeast and could not be confirmed in mammalian cells, the protein revealed interesting physicochemical properties and a peculiar behavior towards standard staining techniques. Localization studies revealed that the protein is expressed in the cytosol as well as in the nucleus.

Comparison of POMP-expression levels in transformed and non-transformed cells showed that malignancy often led to enhanced POMP-expression. Finally, POMP could possibly form tetramers to perform its chaperoning function in 20S proteasome assembly.

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IV D ^EUTSCHE Z USAMMENFASSUNG

Der Komplement Rezeptor 2 (CR2/CD21) wird durch proteolytische Spaltung funktionell reguliert; dieser Vorgang wird als „Ektodomänen Shedding“

bezeichnet und die extrazelluläre Domäne wird als funktionell aktives, lösliches Protein freigesetzt. Durch das experimentelle Entfernen der extrazellulären, membran-nahen Domäne SCR16 (short consensus repeat 16) konnte das Shedding von CD21 unterbunden werden.

In vorliegender Arbeit sollte nun der Einfluss der zytoplasmatischen Domäne auf das CD21-Shedding-Verhalten untersucht werden. Shedding konnte entweder durch die Thiolantioxidantien Glutathion und N-Acetylcystein, oder durch Zell-Aktivierung mittels Pervanadat (ein Tyrosin-spezifischer Phosphatasehemmer), induziert werden. Da Maus B-Zellen, die eine CD21- Mutante ohne zytoplasmatischer Domäne exprimierten, höhere basale und induzierte Shedding-Raten aufwiesen, konnte eine negativ-regulatorische Rolle für diese Domäne bei CD21-Shedding festgestellt werden. Stimulation mit niedrigen (20 μM) bzw. höheren (200 μM) Pervanadat-Konzentrationen führte zu erhöhter CD21 Oberflächen-Expression, bzw. zu CD21-Shedding. Deshalb kann CD21-Shedding als Mechanismus zur Fein-Regulation der B-Zell- Aktivierung vorgeschlagen werden.

Eine weitere Frage war, ob CD21 kontrolliert in der Membran geschnitten würde. RIP (regulated intramembrane proteolysis) findet häufig im Anschluss an Ektodomänen Shedding statt. Tatsächlich konnten in humanen B-Lymphozyten und zahlreichen B-Zell-Linien zwei konstitutiv auftretende, membran-assozierte Carboxy-terminale Fragmente (CTF) nachgewiesen werden (p8^CD21-CTF und p16^CD21-CTF). In humanen T-Lymphozyten, die vermutlich CD21 nicht shedden, konnte keines der beiden Fragmente nachgewiesen werden, obwohl das vollständige CD21 Protein sichtbar war. Interessanterweise konnte in Maus Milzzellen lediglich das kleinere Fragment nachgewiesen werden. Da p8^CD21-CTF selbst in der SCR16-defizienten, Shedding-unfähigen Mutante gefunden wurde, könnte CD21 möglicherweise in einem Shedding-unabhängigen Prozess innerhalb der Membran geschnitten werden.

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In B-Zellen wurde p8^CD21-CTF durch den proteasomalen Weg abgebaut, unabhängig von zusätzlichem Thiolantioxidanzien-induzierten Shedding.

Erstaunlicherweise führte jedoch die Überexpression von CD21 in epithelialen HEK293 Zellen dazu, dass beide CD21-CTFs sowohl durch den lysosomalen als auch durch den proteasomalen Weg abgebaut wurden.

Um intrazelluläre Bindungspartner zu identifizieren, wurde die zytoplasmatische Domäne von CD21 in einem Yeast Two-Hybrid Screening eingesetzt. Ein Kandidat, das proteasomale Reifungsprotein POMP (proteasome maturation protein), wurde identifiziert und intensiver untersucht. Obwohl die Interaktion mit CD21 nur in Hefe, nicht jedoch in Säugerzellen nachgewiesen werden konnte, wies POMP interessante physikochemische Eigenschaften auf und verfügte über spezifische Färbeeigenschaften, wodurch sich das Protein nur sehr schlecht mit Standard Färbemethoden detektieren liess. Lokalisierungsstudien zeigten, dass das Protein sowohl im Zytosol als auch im Nukleus nachweisbar war. Ein Vergleich der POMP-Expression in transformierten und nicht- transformierten Zellen zeigte, dass die maligne Veränderung der Zellen oftmals mit erhöhter POMP-Expression einhergeht. Ausserdem konnte gezeigt werden, dass POMP möglicherweise Tetramere bildet, um seine Chaperon-Funktion beim Zusammenbau der 20S Proteasomen auszuüben.

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V I NTRODUCTION

V.1 Complement receptor type II (CR2/CD21)

The complement system plays a fundamental role in fighting infections and is an important link between innate and adaptive immune responses (1). One central role in bridging these two systems plays the complement receptor type II (CR2/CD21). CD21 belongs to the gene superfamily of regulators of complement activation and is the functional receptor for the complement fragments iC3b and C3d(g) on immune complexes and for the Epstein-Barr virus (EBV) envelope protein gp350 (2,3). The 145 kDa glycoprotein consists of 15 or 16 extracellular short consensus repeats (SCRs) of 60-70 amino acids each, depending on the alternative splicing of exon 11 (4,5), a single spanning transmembrane region and a short cytoplasmic domain of 34 amino acids (aa) (6,7). CD21 is expressed on B-cell lines and on mature B-lymphocytes, but not on early pre-B-cells and later developmental stages after differentiation (8). In addition, CD21 is expressed on follicular dendritic cells (FDCs) (9), and, in lower amounts, on subsets of thymocytes, T-cells (10,11), epithelial cells (e.g. the human embryonic kidney (HEK) 293 cell line) (12), astrocytes (13), mucosal type mast cells (14), and basophilic cells (15). In human pro- and pre-B-cells, CD21-expression is repressed by CpG-methylation in its promotor. Upon B-cell maturation, when CD21 is expressed, CpG-methylation is lost (16,17).

Furthermore, CD21-expression on B-cells and FDCs appears to be regulated by the B-cell activating factor of the TNF-family, BAFF (also known as BLyS, THANK, TALL-1, zTNF4), independent of its function as B-cell survival factor (18). In mice, the complement receptors 1 and 2 (mCR1/2, CD35/CD21) are encoded by the same locus (Cr2) and the two different products are generated by alternative splicing of the mRNA (19).

Functionally, CD21 on B-cells and FDCs is implicated in the recognition and binding of immune complexes while CD21 function in T-cells and all other cell types is not known. In T-cells, the expression of CD21 is developmentally regulated, as only CD4/CD8 double negative thymocytes express membrane bound CD21 (20).

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CD21/CD35-deficient mice reveal decreased antibody production and impaired germinal center (GC) formation in response to T-cell dependent antigens.

Studies with these mice allow the conclusion that CD21 plays a role in negative selection of autoreactive B-cells and in promotion of B-cell differentiation and survival (reviewed in (21)). CD21-functions are exerted by the membrane bound receptor as well as its soluble isoform (sCD21), which derives from proteolytic cleavage of its extracellular domain (“ectodomain shedding”).

V.1.1 CD21 in innate and adaptive immunity

Complement activation is an early response to infection. Additionally to recruiting and activating effector cells in order to phagocytose or lyse the invading antigen, complement cleavage products lead to enhancement of antigen-specific antibody production. The bridging molecule in this process is CD21, which binds to the C3 fragments C3d and C3dg (collectively termed C3d(g)) and iC3b resulting from activation of either classical or alternative complement pathway. The C3d(g)-obsonized antigen binds to its cognate B-cell receptor (BCR), leading to CD21-BCR-crosslinking and activation, proliferation, and differentiation of the B-cell (overview in (22)). On mature B-cells, CD21 forms a non-covalent signal transduction complex in the plasma membrane together with the tetraspanin TAPA-1 (CD81), Leu-13 (CD225) and the pan-B- cell antigen, CD19 (Figure 1). This membrane protein complex, also termed B- cell co-receptor (23-26), enhances BCR signaling in response to complement- coated antigens by several orders of magnitude (27-29). The signal transmitted through the BCR is amplified and the threshold of antigen necessary to initiate cell proliferation is reduced (28,30). Ligation of CD21 results in various signals that are critical for normal B-cell responses (31). Experimental crosslinking of CD21 with C3d(g) or certain anti-CD21 antibodies in the presence of T-cell factors leads to B-cell proliferation and differentiation (32,33). Crosslinking CD21 with membrane IgM promotes T-cell independent proliferation (34,35). In addition, CD21 plays a key role in determining B-cell survival by limiting apoptosis induced by ligation of membrane IgM (36) and by accumulation of the product of the proto-oncogene Bcl-2 (37).

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Figure 1

The B-cell co-receptor complex (adapted from (31)).

CD21 forms a non-covalent complex with CD19 and the two widely expressed CD81 (TAPA1) and CD225 (Leu-13) proteins. Crosslinking of the B-cell receptor (mIgM, drawn with the heterodimer Igα/β) with the co-receptor complex leads to partitioning into lipid rafts, activation of Src-family protein tyrosine kinases (PTKs) and diverse downstream signaling cascades (reviewed e.g. in (31), (19), (22)).

The enhanced signaling of co-receptor crosslinked BCR can be explained by prolonged lipid raft residency of the BCR: The engagement of the BCR alone normally results in sphingolipid- and cholesterol-rich membrane microdomain residency for about 15 min, whilst it was extended to about one hour by co- engagement of the BCR and CD21 mediated by a recombinant antigen-C3d(g) molecule. The co-ligation with the CD21 complex resulted in retardation of degradation and internalization of the BCR and prolonged BCR signaling (30).

For partitioning of the co-receptor complex into signaling active lipid rafts, the tetraspanin CD81 is essential (38).

In the absence of CD21 T-cell dependent immune responses are impaired and the number of GC in the spleen are about 10% of normal (39,40), i.e. CD21 is

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required for normal T-cell dependent responses by internalizing and directing C3d-bound Ag into the MHC class II pathway of B-cells (41-43).

C3 deposition on B-cells may also enhance interaction with CD21 on follicular dendritic cells (FDCs) and vice versa. On FDCs, CD21 plays a major role in rescuing antigen-activated B-cells from apoptosis (44,45), and in promotion of somatic hypermutation (46,47) and class switch (41,48,49).

V.1.2 The role of the cytoplasmic domain of CD21

As discussed above, the important role of CD21 in the BCR co-receptor complex and downstream events is evidenced by the fact that CD21/CD35- deficient mice reveal decreased antibody production and impaired GC formation in response to T-cell dependent antigens (39,40). CD21 physically co-ligates CD19 and the BCR through binding of C3d(g)-tagged antigens and immune complexes (C3d(g)-Ab-Ag) (50). It was shown that the signaling occurs through the long cytoplasmic tail (approximately 240 amino acids) of CD19 by recruiting Lyn, Vav and other Src family protein tyrosine kinases (PTKs) (51,52), phosphatidylinositol 3 (PI-3)-kinase, subsequent generation of inositol-1,4,5- trisphosphate (53) and elevation of intracellular calcium concentrations (54) (Figure 1). A downstream effect of CD19 phosphorylation and PI-3-kinase activation is the recruitment of the serine/threonine kinase Akt (protein kinase B;

PKB) (55,56). Until recently, most data suggested a dominant role of CD19 signaling in this complex (57), but evidence is growing that the cytoplasmic domain of CD21, despite its relatively short length of only 34 (in human, chimpanzee and sheep) or 35 (in mouse and rat) amino acids, plays a pivotal role in cell signaling by itself, independent of CD19 and the BCR (58).

V.1.3 Primary and secondary structure of the cytoplasmic domain of CD21, posttranslational modifications

To date, sequences of the intracellular part of CD21 are available from five species (mouse, rat, sheep, chimpanzee and human). The C-terminal sequence, including short consensus repeat 16 (SCR16), the transmembrane region and the cytoplasmic domain are extremely well conserved within the five mammals (Figure 2).

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P.troglodytes EVNCSSPADMDGIQKGLEPRKMYQYGAVVTLECEDGYMLEGSPQSQCQSDHQWNPPLAVC 60 H.sapiens EVNCSSPADMDGIQKGLEPRKMYQYGAVVTLECEDGYMLEGSPQSQCQSDHQWNPPLAVC 60 O.aries EVNCSFPEHLNGIQNGLEPGRMYQYGAVVTLACEDGYTLEGSPQSQCQEDHRWNPPLAVC 60 M.musculus EVNCSFPEDTNGIQKGFQPGKTYRFGATVTLECEDGYTLEGSPQSQCQDDSQWNPPLALC 60 R.norvegicus EVNCSFPEDTNGIQKGFQPGKTYRFGTTVTLECEDGYSLEGSPQSQCQDENQWNPPLAIC 60

***** * . :***:*::* : *::*:.*** ***** **********.: :******:*

P.troglodytes RS-RSLAPVLCGIAAGLILLTFLIVVTLYMISKHRERNYYTNTSQKE-AFHLETREVYSV 118 H.sapiens RS-RSLAPVLCGIAAGLILLTFLIVITLYVISKHRERNYYTDTSQKE-AFHLEAREVYSV 118 O.aries KSPSSLAPLIAGFSAGVIALFCLGVVTLRMILKHRERNYYTNTNHKE-DVHLEALDIYSA 119 M.musculus KY-RSTIPLICGISVG-SALIILMSVGFCMILKHRESNYYTKTRPKEGALHLETREVYSI 118 R.norvegicus KN-RSTVPLVSGISAG-SAFIILISVIFFIILKSRERNYYTKTRPKDGALHLETREVYAV 118

: * *::.*::.* : * : : :* * ** ****.* *: .***: ::*:

P.troglodytes DPYNPAS 125 H.sapiens DPYNPAS 125

O.aries DPYSPAN 126

M.musculus DPYNPAS 125 R.norvegicus DPYNPAS 125

***.**.

SCR16

TM-1 TM-2 Cyto

***** * . :***:*::* : *::*:.*** ***** **********.: :******:*

: * *::.*::.* : * : : :* * ** ****.* *: .***: ::*:

***.**.

SCR16

TM-1 TM-2 Cyto

***** * . :***:*::* : *::*:.*** ***** **********.: :******:*

: * *::.*::.* : * : : :* * ** ****.* *: .***: ::*:

***.**.

SCR16

TM-1 TM-2 Cyto

Figure 2

Alignment of the amino acid sequences of short consensus repeat 16 (SCR16), transmembrane region 1 and 2 (TM-1 and TM-2), and the cytoplasmic domain (Cyto) of CD21 from chimpanzee (Pan troglodytes, Accession XM_514158), human (Homo sapiens, Accession P20023), sheep (Ovies aries, Accession AAB92375), mouse (Mus musculus, Accession NM_007758) and rat (Rattus norvegicus, Accession XP_213977).

"*" indicate sequence identities, ":" conserved substitutions and "." semi-conserved substitutions. The 7-amino acid dimerization motif (LIxxxxxGVxxxxxxGVxT) is marked (underlined and in bold) in the sheep CD21 transmembrane region (59). Grey shading denotes conserved lysine residues, potential phosphorylation sites in the human sequence are marked with a black closed circle (NetPhos 2.0 (60)), and a potential internalization motif (LHL) is boxed (61). Underlined and in bold in the murine CD21 sequence: Denotation of the two essential tyrosine residues for B-cell antigen internalization and the seven C-terminal amino acids required for efficient antigen presentation (62). Binding sites for p53 (KHRERNYYTD) and for the p68 Ca²⁺-binding protein (KEAFHLEARE) in the human sequence are underlined and in bold (63).

The tertiary structure of the intracellular portion of CD21 is not resolved yet.

Database searches revealed no similarity to other proteins and no obvious functional domains, motifs, or consensus sequences are present within this domain. In none of the known sequences any cysteine residues exist, hence no disulfide bonds can be expected - besides that the reducing environment in the cytosol would not favor this. Furthermore, prediction analysis showed that the intracellular domain of CD21 does not seem to be globular. Secondary structure prediction manifests the presence of two short regions with β-strands and the rest of the sequence shows undefined coiled stretches (64). However, it displays four conserved tyrosines and four less conserved serine/threonine

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residues. In the human sequence three potential phosphorylation sites are present, one tyrosine, one serine and one threonine, as predicted by NetPhos 2.0 (60) (Figure 2). A phosphorylation of CD21 could be shown by stimulation with immobilized anti-µ heavy-chain antibody and/or S. aureus (65), or by the phorbol ester PMA (phorbol-12-myristate-13-acetate) (25,66). Interestingly, CD21 could also be phosphorylated by crosslinking membrane immunoglobulins with anti-IgM antibodies in a cell-free system (67). Hence it will be very important to analyze which of the residues might be phosphorylated, especially considering that different stimuli may phosphorylate distinct sites and could thus connect to diverse signaling pathways.

Other posttranslational modifications of this domain such as ubiquitination were shown in sheep CD21 where two isoforms of CD21 are described: CD21^no (non- ubiquitinated CD21, 150 kDa), structurally similar to known mammalian homologues and CD21^ub (190 kDa), modified by covalent attachment of ubiquitin to the cytoplasmic domain (59,68). CD21^no and CD21^ub form heterodimers on the surface of sheep B-cells, due to a region similar to a rare 7- amino acid non-covalent dimerization motif of α-helices in the transmembrane region. This specific LIxxGVxxGVxxT-sequence was described for the glycophorin receptor (69) and is very similar in sheep CD21 (LIxxxxxGVxxxxxxGVxT), whereas in the transmembrane region of mouse, rat, chimpanzee and human CD21, only aspects of this sequence can be found (Figure 2). In contrast, two lysine residues are highly conserved in the cytoplasmic region in all available CD21 sequences from different mammals, allocating potential sites for ubiquitination (Figure 2). However, the exact function of the ubiquitinated sheep CD21 remains elusive since it is not aimed to degradation but stably expressed in the membrane of sheep B-lymphocytes, non-covalently linked to the conventional form of CD21. The question whether sheep CD21 is mono-, oligo- or poly-ubiquitinated still has to be answered.

Based on the molecular weight of CD21^ub (190 kDa), one could assume it being mono- and/or multi-ubiquitinated, with probably four ubiquitins in total, although only two lysine residues are present in the sheep CD21 cytoplasmic domain and poly-ubiquitination would lead to proteasomal degradation (70) (Figure 2).

Interestingly, attachment of ubiquitin to sheep CD21 correlates with additional

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topography. Peptide mapping, however, revealed a similar peptide core and N- linked glycosylation was comparable in both isoforms. Also, in this study, no association of other cell surface molecules to any of the two isoforms could be demonstrated. In summary, selective ubiquitination could provide a new mechanism to modify CD21 that might play a role in modulation of higher-order structure and/or expression of CD21 during B-cell development. This could contribute to regulate progression of B-cells through the cell cycle and assist in the antigen-induced activation of B-cells for the C3d(g)-tagged antigens. Along with this, ubiquitinated CD21 could be a marker for B-cells at different stages of antigen exposure. The question whether CD21 from other species is ubiquitinated as well is obviously of further interest (59,68).

V.1.4 Interacting partners of the cytoplasmic domain of CD21

The Frade laboratory intensely searched for interacting partners of CD21, especially of the cytoplasmic domain. In some early publications, the interaction with the product of the tumor suppressor gene p53 (71), a p68 Ca²⁺-binding protein (72), and a nuclear p120 ribonucleoprotein (p120RNP) (67) was described. The latter proved to interact with SCR4 rather than with the cytoplasmic tail of CD21, and it could also be shown that the interaction was negatively regulated by phosphorylation of CD21 (63). The extracellular binding site for p120RNP was determined by using various synthetic peptides and conducting blocking experiments; the binding site of p120RNP was described as the 14 amino acid sequence DEGYRLQGPPSSRC within SCR4. In an analogous approach, the binding sites for p53 and p68 were determined to be both within the cytoplasmic domain of CD21 and distinct from each other: the 10 amino acid sequence KHRERNYYTD is the binding site for p53 and the 10 amino acid sequence KEAFHLEARE for p68, respectively (Figure 2). The interaction of CD21 with p53 occurred only in human B-lymphomas and with p68 only in normal, non-transformed B-cells. Other interactions of CD21 have been described with different kinases, such as the protein kinase C, a pp60src- like kinase, and a Ca²⁺/calmodulin dependent kinase (67).

Furthermore, the capacity of CD21 to activate PI-3-kinase and downstream events independently of CD19 was demonstrated by Boullie and co-workers (73) using Raji B-cells and CD21-transfected K-562 cells. CD21 activation

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through monoclonal antibody (BL-13 or OKB-7) crosslinking triggered the interaction of the PI-3-kinase p85 subunit through its Src homology (SH) 2 domains with a tyrosine phosphorylated p95 component, which was later identified as nucleolin (74). In the next step, the downstream activation of the protein kinase B (Akt)/ glycogen synthase kinase-3 (GSK3) pathway was demonstrated, as well as the upstream events of PI-3-kinase, unveiling that nucleolin phosphorylation was performed by pp60src (58). Only recently, the RING (really interesting new gene)-type E3-ligase Cbl (p120) has been shown to be tyrosine phosphorylated upon CD21 activation through pp60src action as well. This triggered the dissociation of Vav from Cbl (75). However, the direct interacting partner(s) of CD21, i.e. the link to pp60src, is still missing. It was previously shown that Cbl modulates BCR-mediated signaling through differential Lyn ubiquitination (76).

A direct approach searching for possible interacting partners of the cytoplasmic domain of CD21 was performed by Gill et al. (77): Using the Yeast Two-Hybrid system to screen a HeLa cDNA library with the cytoplasmic portion with and without the transmembrane region as a bait revealed one promising putative interacting candidate - FHOS/FHOD1 (formin homologue overexpressed in spleen/ formin homology 2 domain containing protein 1), a member of the formin family. The interaction in yeast could be confirmed through co- purification assays and immunofluorescence stainings, where EBV binding to CD21 stimulated plasma membrane aggregation, redistribution, co-localization and clustering of CD21 and FHOS. This effect was greatly diminished by removing the C-terminus of FHOS. Formins or formin homology (FH) proteins are conserved throughout a wide range of species and exert their effects on actin and microtubule networks during meiosis, mitosis, the maintenance of cell polarity, vesicular trafficking, signaling to the nucleus and embryonic development (for review see (78)). A direct involvement of components of the cytoskeleton was previously shown by crosslinking of human CD21 on normal B-lymphocytes through EBV as well as by monoclonal antibodies, which both induced rapid conversion of globular (G-) to filamentous (F-) actin and thus lead to actin polymerization and subsequent activation and transformation of the cells. Preincubation of the cells with botulinum toxin or cytochalasin D, which

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proliferation. These findings indicate that actin rearrangement is crucial for EBV- infection of B-cells (79). Moreover, cells expressing truncated mutants of CD21 lacking the cytoplasmic domain (rCD21) could not be infected with EBV.

Although the intracellular part was not required for C3dg or EBV binding, it was necessary for internalization of cross-linked C3dg as well as for EBV-infection of CD21-transfected murine fibroblast cells and human erythroleukemic K-562 cells (80). Also, a 34 amino acid synthetic peptide corresponding to the CD21 cytoplasmic domain could inhibit B-lymphocyte proliferation triggered by EBV or C3d (81).

V.1.5 CD21 and antigen presentation

Only recently a report demonstrated the importance of specific sequences within the cytoplasmic region of mouse CD21 for antigen internalization and presentation (62). Previous studies have shown that crosslinking of the BCR with the CD19/CD21/CD81/CD225 co-receptor complex greatly enhances antigen presentation to MHC class II-restricted T-cells (28,82), and that human CD21 engagement through complement-tagged antigens induces efficient antigen presentation even without the involvement of the BCR (43,83,84).

Barrault and Knight (62) transfected murine wildtype or various C-terminal truncated CD21 constructs together with cDNAs encoding for a Tetanus toxin C fragment (TTCF)-specific BCR into a CD21/CD35-negative mouse B-cell line, CH27. Crosslinking CD21 with the BCR using (C3d)3-TTCF within this system showed that a tyrosine-based motif was essential and sufficient for internalization of antigens. More precisely, two highly conserved tyrosine residues proximal to the cell membrane, i.e. Y986, Y987 in the murine sequence, account for this effect (see Figure 2). Exchanging Y986 and Y987 to alanine or phenylalanine, respectively, abolished antigen internalization completely. The authors suggest that these tyrosine residues might be a possible target for phosphorylation rather than being structural determinants. In this context it is of note that ubiquitination of a protein at the plasma membrane is often preceded by phosphorylation which creates a docking site for a particular E3 ligase (85). In many cases, serine phosphorylation seems to be essential and the involvement of PEST (proline, glutamic acid, serine and threonine rich) sequences has been discussed, but there are also reports of

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tyrosine phosphorylation playing a role in ligand-induced ubiquitination and subsequent internalization (86), e.g. in the platelet-derived growth factor β- receptor (PDGF receptor) (87). As there are no highly conserved serine residues present in the cytoplasmic domain of CD21 (Figure 2) and no PEST- like sequence can be detected (using the PESTfind program (88,89)), it may well be that the two essential tyrosine residues are important phosphorylation sites for subsequent ubiquitination events and internalization. Interestingly, Barrault and Knight also report that it was essential for internalization to co- crosslink CD21 to the BCR, i.e. the association of the ITAM (immunoreceptor tyrosine-based activation)-motifs containing Igα/β heterodimer with the BCR is important for full internalization capacity. The rate of CD21-internalization was determined to be approximately 2%/min, which is comparable to the intake rates measured for the BCR by Cherukuri and co-workers (30). Meanwhile it is well understood that the cooperation of Igα and Igβ leads to recruitment and activation of signaling molecules required for targeting to MHC II (90).

Additionally, conducting antigen presentation experiments using T-cells specific for TTCF together with the above described B-cell system, Barrault and Knight (62) could show that the seven C-terminal residues of the CD21 sequence are important for antigen presentation. Here, site-directed mutagenesis, exchanging Y1010 to alanine or phenylalanine revealed that this tyrosine residue is not essential. Several motifs and consensus sequences have been described to be important for sorting of transmembrane proteins to lysosomal degradation, or endocytosis. The tight regulation and fine tuning of this sorting process into the right cellular compartment is crucial for normal cell function (reviewed in (85)). A leucine, histidine, leucine (LHL) motif at positions 997 to 999 in the mouse CD21 sequence resembles dileucine-related motifs present in cytoplasmic domains of other receptors involved in either endocytosis or subcellular targeting (61) (Figure 2). Today it is well established that internalization of transmembrane receptors, especially those involved in MHC class II presentation, almost exclusively occurs via the lysosomal pathway (reviewed in (91)). In fact, in another important study by Hess and co-workers (92), C3dg- coated colloidal gold particles where traced by electron microscopy in the B- lymphoblastoid cell line, Raji. Binding of the C3dg-gold complex was analyzed

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to be located to microvilli and internalization occurred via coated pits to different compartments of the endocytic pathway. The exact mechanism of this phenomenon, and whether clathrin is involved in the uptake process, is not resolved yet. After 120 minutes, more than 70% of the C3dg-coated gold particles could be detected in endosomal or lysosomal compartments in which a co-localization with MHC class II was shown through HLA-DR (human leukocyte antigen-DR) co-staining. Preincubation with a monoclonal antibody (mAb FE8) abolished C3dg-gold binding and internalization, whereas a control CD21- antibody which is not able to block the C3dg binding site (mAb HB5) did not abrogate the uptake (92).

Last but not least, CD21 plays a direct role in antigen presentation in the GC reaction. Here, FDCs present Ag-Ab complexes independent of MHC through Fc receptors such as CD23 (FcεRII) and CD32 (FcγRII), or through complement receptors such as CD21 and CD35 over a long time period and in a very efficient way, in order to activate B-cells to proliferate and differentiate into plasma and memory B-cells (reviewed in (93)). The exact mechanism how this long-term presentation is achieved and how CD21 exerts its specific tasks in this process has yet to be determined.

In summary, the cytoplasmic tail of CD21 does play an important role for normal B-cell function, the 34 to 35 amino acids are not only required for antigen internalization and presentation, but also for the execution of diverse signaling functions.

As CD21 is also present as biological active soluble molecule (sCD21), many of its actions may be performed by this isoform.

V.1.6 Soluble CD21

CD21 is one of several cell surface proteins that undergoes regulated proteolytic cleavage of the extracellular domain, which results in the release of the ectodomain as a soluble and biologically active protein. This phenomenon is termed “ectodomain shedding” (94). The soluble form of CD21 (sCD21) is found in human plasma and in cell culture supernatants of lymphoid cells (95-97).

Mass spectrometric studies revealed that sCD21 in human plasma of healthy donors might consist predominantly of the short form of CD21 without the exon 11 encoded sequence. The N-terminus of sCD21 lacked the leader

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peptide and the C-terminus was truncated. Therefore it was concluded that only the extracellular portion of CD21 is shed and that shedding takes place adjacent to the plasma membrane within SCR16. The exact cleavage site(s) however, is unknown so far (98,99). Several groups have aimed at identifying the exact molecular weight of sCD21 isolated from human plasma and of cell culture supernatants of lymphoid cells, respectively. Using affinity purification and discontinuous high-speed density gradient centrifugation, a single species of sCD21 from human plasma with a molecular weight of 126 kDa was isolated (98). However, others found several sCD21 proteins with different molecular weights. Fremeaux-Bacchi and colleagues (100) purified sCD21 from human sera and identified a major 135 kDa and a minor 90 kDa sCD21 band, while in Raji cell culture supernatants only a single sCD21 band of 135 kDa was detected. Autoradiographs of affinity-purified and ³⁵S-labeled sCD21 from a B- lymphoblastoid cell line (B-LCL) revealed a major sCD21 band at 130 kDa (101). Taken together, it appears that CD21 contains several alternative cleavage sites as also described e.g. for the transferrin receptor or the angiotensin-converting enzyme (ACE), but with a preference for one cleavage product (102,103). Unfortunately, the protease(s) responsible for CD21- shedding also have not been identified yet.

V.1.7 Regulation of CD21-shedding

The phorbol ester phorbol-12-myristate-13-acetate (PMA) is an agent commonly used for activating shedding of a variety of structurally and functionally unrelated cell surface proteins, such as tumor necrosis factor-α receptor (TNF-α receptor), L-selectin, or amyloid precursor protein (APP) and many more (104). PMA activates a signal transduction pathway that includes activation of protein kinase C (PKC) (105). Stimulation of Raji B- or primary B- cells with PMA also induced CD21-shedding, and the effect was enhanced by additional stimulation with the Ca²⁺-ionophore ionomycin. Ionomycin makes Ca²⁺-ions available for the PKC and therefore amplifies PMA-effects (99).

Besides, PMA acts as a mitogen by mimicking signal transduction through the BCR and thus stimulates B-cell activation. CD21-shedding could therefore also be achieved in B-cells by stimulating B-cell differentiation through crosslinking

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interleukin (IL-) 2 (99). The tyrosine-specific phosphatase inhibitor pervanadate (PV) mimics BCR signaling and it was shown in several studies that PV induces protein tyrosine phosphorylation and cell activation (106). It was demonstrated previously that shedding of human CD21 may be a redox-regulated process and can be induced with the thiol antioxidants glutathione (GSH), N- acetylcysteine (NAC), the antioxidant β-mercaptoethanol, and the oxidant PV in CD21-transfected HEK293 cells, peripheral blood mononuclear cells (PBMC) and the Burkitt´s lymphoma line, Daudi (107). Naturally occurring oxidants such as H2O2 are connected to lymphocyte activation and play a crucial role as a secondary messenger in the initiation and amplification of signaling of the antigen receptor (108).

Interestingly, CD21 mutants lacking the extracellular membrane adjacent SCR16 revealed abolished basal and induced shedding rates (107). It was concluded that NAC and GSH may act directly on CD21 and/or a metalloprotease and in contrast to that, PMA and PV operate through intracellular signaling, since PKC-inhibition subdued sCD21 formation in PV- and PMA- but not in NAC- or GSH-stimulated cells (107).

Redox-regulation is important for multiple cellular functions, signal transduction and gene expression, especially in immune cells (109,110). The cellular redox potential is mainly regulated by the thioredoxin- and GSH-systems and the GSH:GSSG (glutathione-to-glutathione disulfide) ratio is an indicator for the cellular redox state (111,112). GSH is a ubiquitous thiol-containing tripeptide (γ- Glu-Cys-Gly) that plays important roles in antioxidant defense, and modulates gene expression and apoptosis (110). Its concentration in plasma is relatively low (about 2-20 μM) and higher within the cell (0.5-10 mM), where 85-90% is present in the cytosol (112). However, GSH is not transported efficiently into cells. In contrast, NAC is readily deacetylated in cells to yield the rate-limiting factor for GSH-synthesis, L-cysteine, and can thus be regarded as GSH- precursor (113). NAC-stimulation was shown to inhibit IL-4 synthesis from T- cells, and to down-regulate IgE production (by decreasing the mature ε messenger RNA), and CD21-expression on B-cells. Furthermore, expression of CD40 was reduced to 50%, while CD19 and CD20 were not affected (114).

Also, EBV-infection can be inhibited in vitro by NAC as a result of down- regulating CD21 protein from the cell surface. This was independent of

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intracellular GSH levels, i.e. the involvement of a GSH-dependent signaling pathway could be ruled out by using an inhibitor of GSH-synthesis (115).

Consequently, NAC has been suggested as treatment for HIV, EBV and other viral infections (109,114-116).

V.1.8 Functional consequences of CD21-shedding

Any increment in sCD21 amounts due to enhanced CD21-shedding could competitively inhibit the binding of CD21 ligands that activate or influence the neighboring environment. Thus, changes in sCD21 levels could contribute to the fine-regulation of CD21-dependent responses and B-cell activation. The most likely ligands of sCD21 in a local environment such as lymph nodes or GCs, are iC3b, C3d(g) and CD23, the low affinity receptor for IgE. sCD21 was shown to circulate in plasma in complexes with a trimeric form of soluble CD23 (sCD23) (100) and an interaction of sCD23 with membrane bound CD21 of IL-4 stimulated B-cells induced IgE-synthesis (117). The sCD23-induced IgE- synthesis could be inhibited by a preceding incubation with sCD21, thereby inhibiting binding of sCD23 to cell surface CD21. Taken these findings together, a role for sCD21 in regulating IgE-synthesis in vivo is conceivable (100,118).

Fremeaux-Bacchi and co-workers (118) showed that sCD21 also serves as functional ligand for CD23 on monocytes and that binding of sCD21 to CD23 induces cellular responses in monocytes that are characteristic for CD23 ligation. By addition of sCD21 to IL-4-stimulated CD23⁺ monocytes, intracellular cGMP accumulation as well as increased IL-6 and TNF-α secretion were measured. sCD21 also influenced cell surface expression of certain monocyte antigens important for antigen presentation or interaction with other leukocytes such as increased expression of CD40 and the MHC class II molecule HLA-DR, as well as decreased expression of CD14. Hence, sCD21 displays a cytokine- like activity in modulating monocytes functions (118).

Hebell and co-workers (119) used recombinant soluble fusion proteins of the C3-binding domain of CD21 and immunoglobulin G1 to suppress the antibody response to a T-cell dependent antigen in mice, suggesting a new possibility for humoral immunosuppression. Inhibition of B-cell/FDC interactions by sCD21 was shown to reduce the antibody response 10- to 1000-fold (120). As CD21 is

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by preincubation of PBMC with recombinant full length or truncated chimeric sCD21 proteins. Together with the findings that sCD21 levels are elevated in EBV-associated malignancies (95), this implies a chief role for sCD21 as therapeutic agent against EBV-infections in humans (121,122).

In summary, these data show that sCD21 is a functional active molecule with ligand-binding properties similar to that of membrane-bound CD21. This supports the suggestion that the amount of sCD21 in plasma could be a modulator of immunity.

V.1.9 CD21 and sCD21 in disease

CD21 has been associated to a number of diseases, especially to EBV-related ailments, as CD21 serves as its receptor, and to autoimmune diseases. sCD21 levels are often altered in pathologic conditions including various lymphoproliferative leukemias, such as B-CLL (B cell-type chronic lymphocytic leukemia) (123), acute EBV-infection and other virus-associated diseases (95,124), and autoimmune diseases (125,126). However, the precise function of sCD21 in the respective diseases remains to be investigated.

EBV is the best studied member of the herpes virus family and is involved in the pathogenesis of several human malignancies, as endemic Burkitt´s lymphoma, nasopharyngeal carcinoma, Hodgkin’s lymphoma, polyclonal lymphomas in immuno-compromised individuals and many more (127). sCD21 has been described as a marker of B-cell activation in humans and elevated sCD21 levels were found in patients with EBV-associated malignancies. Furthermore, sCD21 can inhibit EBV-binding and infection of B-cells (95,122). However, as sCD21 is able to bind its ligands in plasma, the increased amounts of sCD21 caused by EBV-infection may contribute to the immuno-regulatory dysfunctions observed in EBV-associated diseases (124). Also, CD21 and its soluble form (sCD21) have been described to bind to the low-affinity IgE-receptor CD23 and to sCD23, thereby modulating IgE responses and monocyte activation and differentiation. Therefore, an imbalance of sCD21 and sCD23 may contribute to the development of allergic reactions (100,118).

The mechanism by which CD21 might regulate B-cell reactivity to autoantigens has not been clarified yet. It may involve direct effects on B-cell tolerance or indirect effects on T-cell tolerance (128). The involvement of CD21/CD35 in the

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control of self tolerance was shown by Prodeus et al. (129), where CD21/CD35- deficient mice where crossed in mice expressing transgenic anti-hen-egg lysozyme (HEL) specific antibodies (130). In the presence of both HEL in the blood and anti-HEL transgenic B-cells, B-cells were rendered unresponsive (anergic) in the periphery. CD21-deficient B-cells were able to up-regulate CD86 and the BCR, but still did not respond with antibody secretion. Hence, CD21 has some role in the induction of anergy. Crossing CD21/CD35-deficient mice with Fas-deficient mice that normally develop autoimmunity of a lupus-like type, renders the offspring with higher anti-DNA autoantibody titers, and thus enhanced autoimmunity (129). Furthermore, CD21-expression has been linked to enhanced susceptibility for systemic lupus erythematosus (SLE) (131). As BAFF independently regulates CD21- and CD23-expression (18), and BAFF overexpression leads to autoantibody production, seen in SLE, primary Sjögren`s syndrome (pSS) and rheumatoid arthritis (RA), some close link between over-activation of B-cells, CD21-expression and the development of autoimmunity may be drawn. Moreover, treatment of SLE mice with a BAFF protein antagonist ameliorates disease progression and enhances survival (132). In patients with SLE, an autoimmune disease characterized by antibodies specific for nuclear antigens such as dsDNA, sCD21 levels are reduced (125).

Low sCD21 levels are also found in the Sjögren`s syndrome, a disease characterized by autoantibodies directed against salivary and lacrimal glands. In juvenile arthritis, though, sCD21 levels were not altered (126). These data together with data from patients with autoimmune rheumatoid arthritis having reduced expression levels of CD21 in synovial B- and T-cells (133) and significantly lower sCD21 plasma levels (126), indicate a role for CD21 in autoimmunity. Interestingly, in SLE, pSS and RA, sCD23 levels have been described to be up-regulated (132).

To further understand the regulation and specific mechanism of CD21- shedding, proteases known to be involved in shedding of other proteins must be considered as potential CD21-sheddases.

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V.2 Ectodomain shedding and involved proteases

Many functionally and structurally unrelated cell surface proteins such as growth factors, growth factor receptors, ectoenzymes, precursors of cytokines or cell adhesion molecules release their extracellular domain (ectodomain) as soluble molecules into the extracellular space by a highly regulated, proteolytic process termed “ectodomain shedding”. It leads to a decrease of cell surface proteins and at the same time to an increase of the soluble forms of these proteins. It has been estimated that 2-4% of all cell surface proteins are shed. Importantly, the soluble forms of the respective proteins retain their biological activity and have regulatory functions (reviewed e.g. (104,134-136)).

Only recently, several protease families have been added (137) to the family of metalloproteases that are potentially implicated in the liberation of soluble ectodomains of cell surface proteins. These include aspartyl proteases (138), cysteine proteases (such as cathepsins) and serine proteases (e.g. elastase, coagulation factors, tissue plasminogen activator, plasmin, urokinase plasminogen activator) (137).

V.2.1 Matrix metalloproteases and ADAMs

Numerous proteases that cleave the ectodomain of cell surface molecules have been found to belong to the family of metalloproteases (MPs) (134).

Metalloproteases include the secreted MMPs (matrix MPs) and ADAMTS´ (a disintegrin and metalloproteinase with thrombospondin motifs), and the transmembrane or membrane-type metalloproteases (MT-MPs), like the membrane-type matrix metalloprotease (MT-MMP) and the ADAM (a disintegrin and metalloprotease) families (139,140).

Metalloproteases are Zn²⁺-dependent endopeptidases involved in extracellular matrix (ECM) remodeling and degradation (141). Under normal physiological conditions, their activities are accurately regulated at the level of transcription, activation of the precursor zymogens and by inhibition with endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Alterations in the regulation of MP activity play a central role in many pathologic situations such

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as tumor growth, invasion and metastasis as well as fibrosis, arthritis and atherosclerosis (139,140,142,143).

Actually, ADAM metalloproteinase disintegrins have emerged as the major proteinase family that mediates ectodomain shedding (144). The best studied example known to cleave a plethora of substrates upon induction is the TNF-α converting enzyme (TACE/ADAM17). Besides pro-TNF-α, TACE/ADAM17 also cleaves the hyaluronic acid receptor CD44, APP, human epithelial growth factor receptor HER-4, Notch, L-Selectin, only to name a few (145). Shedding of TACE/ADAM17 substrates can be stimulated by the phorbol ester PMA as well as by the phosphatase-inhibitor PV (135). The mechanism and physiological relevance of these kinds of TACE/ADAM17 activation, however, remain to be determined. Only recently a role for bacteria as physiological activators of TACE/ADAM17-expression has been established in airway epithelial cells (146). TACE/ADAM17-mediated cleavage is very fast, the t1/2 for this process has been described to be 5-10 min and the cells virtually become devoid of ectodomains within this time (134). For basal or uninduced shedding of certain TACE/ADAM17 substrates, however, ADAM10 (147) and not yet identified proteases have been shown to be involved (134). At present very little is known about the regulation of other ADAMs than TACE/ADAM17 (135). Only recently, several MT-MMPs have also been shown to cleave some TACE/ADAM17 substrates, e.g. CD44 is not only cleaved by ADAM10 and TACE/ADAM17, but also by MT1-MMP (148). MT1-, MT2-, and MT3-MMP may also be liable for betaglycan cleavage (149).

Many metalloproteases can be activated by oxidation and dissociation of the cysteine thiol-zinc linkage from a latent enzymatic site. This complex is referred to as a “cysteine zinc switch” mechanism (150). For example, nitric oxide (NO), a molecule produced in a variety of inflammatory conditions, was shown to regulate TACE/ADAM17-activity and ectodomain shedding (151).

V.2.2 BACE, the beta-site amyloid precursor protein secretase

Alzheimer's disease (AD) is a chronically progressive neurodegenerative disease. The key protein in the pathophysiology of AD is the ubiquitous receptor-like molecule APP, whose proteolytic processing can generate highly

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of plaques detected in the brain parenchyma of AD patients (152). The β-site APP cleaving enzyme (BACE1) is a transmembrane aspartic protease of the pepsin family with β-secretase activity. It performs the first proteolytic step in the processing of APP to Aβ. After BACE1 cleavage 28 aa N-terminal from the transmembrane domain, soluble APPβ is released and APP becomes a substrate for γ-secretase (see also IV.3.1). This leads to the liberation of Aβ, which accumulates in the disease-characteristic senile plaques in AD (Figure 6) (153). BACE1 itself is processed by furin proteolysis in the Golgi-network, its ectodomain is shed by ADAM10, and it is internalized due to an essential dileucine motif and degraded by the lysosomal pathway (154). The regulation of BACE1 activity is still under investigation; it does not possess high sequence specificity, but has a preference for hydrophobic residues, especially of leucines, at the P1 position (155). BACE1 clusters in lipid rafts, and this is also where APP cleavage occurs preferentially. For this, APP and BACE1 are both internalized and meet in endosomes where the BACE1 pH-optimum prevails (153). Furthermore, an involvement of heparan-sulfate proteoglycans (HSPGs) and heparansulfate (HS) has been implicated in the negative regulation of BACE activity (156).

BACE2, highly homologous to BACE1, was also shown to process APP, but it has a different tissue distribution and thus does not contribute to the amyloidogenic pathway (153). 40% of BACE1-deficient mice and 60% of the double-knock-out mice die very soon after birth, probably due to deficient immune responses, since the high mortality rate was absent in mice housed in pathogen-free facilities (157).

At present, only few BACE-substrates besides APP have been identified:

BACE1 also cleaves β-subunits of voltage-gated sodium channels (158), the Golgi-resident sialyltransferase ST6Gal I (159), the low density lipoprotein receptor-related protein (LRP) (160), and P-selectin glycoprotein ligand-1 (PSGL-1), which confers leukocyte adhesion to endothelial cells (155).

V.2.3 Serine proteases involved in shedding

Another group of proteases potentially involved in ectodomain shedding is the well conserved family of membrane-anchored serine proteases. Chymotrypsin and trypsin are the prototype members of this large family. These enzymes are

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involved in a multitude of cellular functions including blood coagulation, wound healing, digestion and immune responses. They have been implicated in the pathogenesis of several diseases, such as tumor growth, invasion and metastasis. The physiological role of most membrane-anchored serine proteases, however, is unclear and studies are just beginning to identify specific endogenous targets (161,162). Moreover, soluble serine proteases as thrombin and plasmin, e.g. have been shown to cleave substrates like CD40L (163), E- cadherin (164), L1 (165), syndecan-1 and -4, IL-8, and heparan sulfate (166,167). It is of note, that extensive crosstalk between the different proteolytic systems has been documented (162).

The mammalian subtilisin/Kex2p-like serine endoproteases are also implicated in a multitude of biological processes, including cell proliferation, motility, and adhesion (168,169). They represent a family of pro-protein convertases (PC family) that process precursors of peptide hormones, neuropeptides, MMPs and many other proteins into their biologically active forms (169-171). Currently about nine different PCs are known (furin/PACE, PC1/PC3, PC2, PC4, PACE4, PC5/PC6, PC7/PC8/LPC, SKI/S1P, and NARC-1/PCSK9), but the best-studied member is furin (169). It is ubiquitously expressed, and mainly localizes to the trans-Golgi compartment. Furin is a membrane-associated Ca²⁺-dependent endoprotease that converts pro-proteins in the constitutive secretory pathway.

Chelators like EGTA and EDTA strongly inhibit its activity and for optimal activity, furin requires 1-2 mM Ca²⁺ (171). It is well known to activate several MMPs (172) and ADAMs, such as ADAM10 and TACE/ADAM17, which are activated together with PC7, another PC-family member (173). The furin- consensus cleavage sequence R-X-(K/R)-R is important for substrate recognition. Furin is e.g. also responsible for the first Notch cleavage (174) (Figure 5).

Technically, the rhomboid family of membrane-anchored serine proteases, with the Drosophila rhomboids and their vertebrate counterparts, the RHBDLs, are also sheddases, because they release soluble ectodomains (for review see (175)), but as they also cleave their substrates within the membrane, they will be discussed in the next section on intramembrane cleaving proteases.

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V.3 Intramembrane cleaving proteases (I-CLiPs) and regulated intramembrane proteolysis (RIP)

Recently, a novel mechanism for signal transduction has been discovered, termed regulated intramembrane proteolysis (RIP) (176) (Figure 3). The key feature of RIP is that substrates are cleaved within the membrane, a process formerly thought to be biochemically impossible. Only special proteases are able to hydrolyze peptide bonds within the hydrophobic environment of the membrane and are consequently termed intramembrane cleaving proteases (I- CLiPs) (177,178).

I-CLiPs such as the metalloprotease-type site 2 protease (S2P), the γ-secretase complex with aspartyl-like activity originating from presenilin (179) and another aspartic protease, the signal peptide peptidase (SPP) (180), hydrolyze peptide bonds within the transmembrane domain (TMD) of signaling molecules such as SREBP, Notch, and the non-classical MHC class Ib molecule HLA-E, respectively. All three enzymes require a prior cleavage at the juxtamembrane region by another protease. In contrast to that, the serine-type protease rhomboids are able to cleave their substrates in the membrane without prior processing (175). In vertebrates, only presenilin and the rhomboid-like proteases cleave type I membrane proteins, and type II cell surface proteins are cleaved by SPP and S2P (177). Only the γ-secretase complex is able to cleave, apparently sequence-independent, a huge variety of substrates, while the other I-CLiPs show some sequence specificity towards their substrates (175).

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Figure 3

Many cell surface molecules sequentially undergo ectodomain shedding and regulated intramembrane cleavage (RIP) (adapted from (145)).

CTF = carboxy-terminal fragment, ICD = intracellular cytoplasmic domain

It has been demonstrated for several type I membrane proteins that a second or third cleavage step occurs within the membrane after the initial cleavage step by a metalloprotease or ADAM. To release the intracellular cytoplasmic domain (ICD) by presenilin, the membrane-tethered, carboxy-terminal fragment (CTF) must be recognized by nicastrin, which is another element of the γ-secretase complex (Figure 4A). This is well established for Notch, APP, Erb-B4, CD44 and many more that are cleaved by the γ-secretase complex (181). The γ-secretase complex was described to be active in the compartment distal to the ER-Golgi (182).

Upon their release, ICDs have been shown to move to the nucleus, where they can regulate transcription of specific genes. Non-nuclear signaling has also been reported from cytosolic fragments, e.g. of E-cadherin, p75NTR and N- syndecan (181).

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Figure 4

The γ-secretase complex (from (183)) and rhomboid-1 (from (175)).

(A) Presenilin is the active aspartic-type protease within the γ-secretase complex and nicastrin serves to identify cleavable targets after their extracellular domains are shed through site-specific proteolysis, by recognizing the amino-terminal formed stubs (184).

(B) Drosophila Spitz was the first rhomboid substrate to be identified; the epidermal growth factor (EGF) domain of Spitz is released into the lumen upon the constitutive cleavage in the N-terminal region of the TMD by rhomboid-1.

V.3.1 The γ-secretase complex

Here, two examples of γ-secretase substrates shall be discussed in further detail, Notch (Figure 5) and APP (Figure 6), because the several protease cleavage steps they both undergo are well-studied, yet partly different. The type 1 transmembrane receptor Notch undergoes a first cleavage (S1) in the trans-Golgi compartment by a furin-convertase, but the two Notch parts remain associated after cleavage and are delivered together to the cell surface. The second cleavage (S2) is performed by TACE/ADAM17 or ADAM10, and the third step (S3) in Notch processing is the intramembrane cleavage step. It has recently been shown that Notch needs to be mono-ubiquitinated and internalized by clathrin-dependent endocytosis, prior cleavage by the γ- secretase complex (174).

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Figure 5

Notch signaling involves RIP (adapted from (185)).

Notch is processed by at least three different proteases: First, by a furin-type convertase (S1) in the trans-Golgi network before being transported to the cell membrane as functional receptor. Second, at the cell surface, ADAM10 or TACE/ADAM17 cleave Notch (S2) after activation, e.g. by ligand binding (Delta, or Serrate), leading to the release of soluble Notch (NECD; Notch extracellular domain).

Finally, the membrane-tethered, carboxy-terminal fragment (NEXT, Notch-CTF) is cleaved by the γ-secretase complex (S3) to release the intracellular domain of Notch (NICD) which proceeds to the nucleus and acts as transcriptional regulator.

For APP two major processing pathways exist, the amyloidogenic and the antiamyloidogenic (Figure 6). APP is first cleaved by either α- or β-secretase, to release soluble APPα or APPβ, and to form the membrane-associated APP- CTFα (C83) or APP-CTFβ (C99), respectively. These are substrates for the next cleavage by γ-secretase, liberating p3, or the Aβ peptide(s), respectively, which may - due to different cleavage sites - consist of different lengths (Aβ37- Aβ43). Different mutations in APP and presenilin lead to an increase of Alzheimer’s disease specific Aβ42 peptides. These γ-secretase cleavages occur approximately in the middle of the transmembrane domain (TMD) of APP, rendering the CTFs membrane-associated. Another γ-secretase cleavage can take place at the ε-site close to the cytoplasmic site of the TMD, thus liberating the intracellular domain of APP (termed AICD, γAPP-ICD, or CTFγ). AICD

Functional analysis of the cytoplasmic domain of complement receptor type II (CR2/CD21)