j ourna l ho me page: www.elsev ier .com/ locate / immlet
CIR interacts with ligands from both endogenous andathogenic origin
arien Bloema,b,1, Ilona M. Vuista, Meike van den Berka, Elsenoor J. Klavera,rma van Diea, Léon M.J. Knippelsb,c, Johan Garssenb,c, Juan J. García-Vallejoa,2,andra J. van Vlieta,2, Yvette van Kooyka,∗
Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The NetherlandsDanone Research, Centre for Specialized Nutrition, Wageningen, The NetherlandsDepartment of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University,he Netherlands
r t i c l e i n f o
rticle history:eceived 29 July 2013eceived in revised form 13 October 2013ccepted 5 November 2013vailable online 14 November 2013
eywords:-type lectinsendritic celllycanlycosylationathogen and cancer
a b s t r a c t
C-type lectins on dendritic cells function as antigen uptake and signaling receptors, thereby influ-encing cellular immune responses. Dendritic cell-specific intercellular adhesion molecule-3-grabbingnon-integrin (DC-SIGN) is one of the best-studied C-type lectin receptors expressed on DCs and its gly-can specificity and functional requirements for ligand binding have been intensively investigated. Thecarbohydrate specificity of dendritic cell immunoreceptor (DCIR), another DC-expressed lectin, was stilldebated, but we have recently confirmed DCIR as mannose/fucose-binding lectin. Since DC-SIGN andDCIR may potentially share ligands, we set out to elucidate the interaction of DCIR with established DC-SIGN-binding ligands, by comparing the carbohydrate specificity of DCIR and DC-SIGN in more detail. Ourresults clearly demonstrate that DC-SIGN has a broader glycan specificity compared to DCIR, which inter-acts only with mannotriose, sulfo-Lewisa, Lewisb and Lewisa. While most of the tested DC-SIGN ligandsbound DCIR as well, Candida albicans and some glycoproteins on some cancer cell lines were identified
as DC-SIGN-specific ligands. Interestingly, DCIR strongly bound human immunodeficiency virus type 1(HIV-1) gp140 glycoproteins, while its interaction with the well-studied DC-SIGN-binding HIV-1 ligandgp120 was much weaker. Furthermore, DCIR-specific ligands were detected on keratinocytes. Further-more, the interaction of DCIR with its ligands was strongly influenced by the glycosylation of DCIR. Inconclusion, we show that sulfo-Lewisa is a high affinity ligand for DCIR and that DCIR interacts withligands from both pathogenic and endogenous origin of which most are shared by DC-SIGN.
ntercellular adhesion molecule-3-grabbing non-integrin; DCIR, dendritic cellmmunoreceptor; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine;CV, Hepatitis C Virus; HIV-1, human immunodeficiency virus type 1; ITIM,
mmunoreceptor tyrosine-based inhibitory motif; Methyl-Man/Glc, Methyl-�--glycopyranoside/Methyl-�-mannopyranoside; MHC, Major Histocompatibilityomplex; PAA, polyacrylamide; PAMP, pathogen associated molecular pattern; SP,oluble products; TLR, Toll-like receptor; TMB, 3,3′ ,5,5′-tetramethylbenzidine.∗ Corresponding author at: Department of Molecular Cell Biology and Immunol-gy, VU University Medical Center, P.O. Box 7057, 1007MB Amsterdam, Theetherlands. Tel.: +31 20 4448080; fax: +31 20 4448081.
E-mail address: [email protected] (Y. van Kooyk).1 Present address: Laboratory for Monoclonal Therapeutics, Sanquin Diagnostics,msterdam, The Netherlands.2 These authors contributed equally to this work.
Dendritic cells (DCs) play a critical role in shaping innate andadaptive immune responses. DCs are equipped with differentreceptor families, such as the Toll-like receptor (TLR) family thatenables them to respond to pathogen associated molecular pat-terns (PAMPs) and danger associated molecular patterns (DAMPs)[1]. TLR stimulation will induce DC maturation, characterized bythe upregulation of co-stimulatory molecules, the secretion ofcytokines and the migration of DCs to the lymph nodes [2,3]. Otherreceptors, like the C-type lectin receptors (CLRs), are involved inantigen internalization and the routing to Major Histocompatibil-ity complex (MHC) class I and II loading compartments, needed forMHC class I and II-dependent T cell activation [4,5]. In addition,
some CLRs are capable of modulating TLR-induced responses byaltering intracellular signaling pathways [6,7].
CLRs recognize glycan structures present on pathogens, com-mensals and self-glycoproteins [7]. The occurrence of a Glutamine
cid-Proline-Asparagine (EPN) motif inside the carbohydrateecognition domain (CRD) predicts fucose and mannose binding,hile the Glutamine-Proline-Aspartic acid (QPD) motif is involved
n binding to galactose- or N-acetylgalactosamine (GalNAc)-ontaining glycans [8]. Dendritic cell immunoreceptor (DCIR)ontains an unusual sequence in the putative CRD, whereby thesparagine residue is replaced by a serine residue, resulting inn EPS motif [9]. This receptor is the only classical CLR that con-ains an intracellular immunoreceptor tyrosine-based inhibitory
otif (ITIM). The glycan specificity of this lectin has only recentlyeen investigated, however with conflicting results, as both man-ose/fucose specificity [10] as well as binding to sulfated LacNAcnd Lac, and biantennary N-glycans [11] has been reported. Weave elucidated the glycan specificity of DCIR as well and our resultslearly demonstrate DCIR binding to mannotriose and Lewisb [12],onfirming DCIR as fucose/mannose-binding lectin.
DC-specific intercellular adhesion molecule-3 (ICAM-3)-rabbing non-integrin (DC-SIGN) is another fucose/mannoseinding lectin expressed on DCs [13]. In contrast, DCIR is expressedn a variety of immune cells besides DCs, including monocytes,eutrophils, B-cells and activated T cells [9,14–16]. Comparedo DCIR, the glycan specificity of DC-SIGN has been extensivelytudied, demonstrating DC-SIGN recognition of high mannose andnsialylated Lewis structures [17]. The interaction of DC-SIGNith fucose-containing glycans is of higher affinity than theC-SIGN-mannose interaction [10].
Based on its glycan specificity, DC-SIGN binding to variousndogenous and exogenous ligands has been suggested [17,18].fter the initial report on the binding of DC-SIGN to human
mmunodeficiency virus type 1 (HIV-1) gp120 [19], many otherathogens have been described as DC-SIGN ligands, including fungi,uch as Candida albicans [20], viruses, like Hepatitis C Virus (HCV)21,22], bacteria, such as Helicobacter pylori [17] and Mycobacteriumuberculosis [23] and helminths, like Schistosoma mansoni [24]. Inddition, various endogenous proteins have been reported as DC-IGN ligands, including the adhesion molecules ICAM-3, ICAM-2,EACAM1 and Mac-1 [13,19,25–27]. Altered cellular glycosylationuring oncogenesis results in the expression of DC-SIGN-bindinglycans on tumor associated antigens, such as CEA [28,29].
Only recently the first DCIR-binding ligand, HCV, has beeneported [30]. Furthermore, HIV-1 infectivity has been shown toe dependent on DCIR-induced signaling [31,32], however actualinding of HIV-1 to DCIR has not been demonstrated yet. The recog-ition of endogenous ligands by DCIR still needs to be established.
The potential overlapping glycan specificities of DC-SIGN andCIR prompted us to investigate this in more detail, particularly asoth are expressed simultaneously on DCs and the functional out-ome might depend on the combined interaction of antigens withoth lectins. Since DCIR and DC-SIGN most likely have distinct func-ional properties, we set out to investigate the glycan specificity ofCIR in more detail, thereby exploring whether DCIR was able to
nteract with known DC-SIGN-binding ligands. We here show thatulfo-Lewisa is a high affinity ligand for DCIR and that DCIR interactsith ligands from both pathogenic and endogenous origin.
. Materials and methods
.1. Antibodies, Fc chimeric proteins, lectins, glycans and ligands
DCIR-Fc consists of the extracellular domains of DCIR (aminocid residues 208–689) fused at the C terminus to the Fc domain
f human IgG1 [33] and was produced in Chinese Hamster OvaryCHO) cells (DCIR-Fc) and in CHO Lec8 cells (DCIR-Fc Lec8) [34].C-SIGN-Fc was produced as described previously [35]. All Fconstructs were purified using Hi Trap Protein A HP columns (GE
etters 158 (2014) 33–41
Healthcare Europe). Human IgG1 kappa (Serotec) was used as acontrol Fc protein. Antibodies used are �-Lewisa (clone T174),�-Lewisb (clone T218), �-LewisX (clone P12) and �-LewisY (cloneF3) (Calbiochem), goat-�-mouse-IgM-PO (Nordic ImmunologicLaboratories), goat-�-mouse-IgM-FITC (Jackson Immunore-search), goat-�-mouse-Ig-PO (Dako), goat �-mouse IgG AlexaFluor 488 F(ab′)2 (Invitrogen) and goat-�-human Fc-PO (JacksonImmunoresearch). Other reagents include streptavidin-AlexaFluor 488 (Invitrogen), streptavidin-PO (Invitrogen), biotinylatedlectin Concanavalin A (ConA, Vector Labs) and Methyl-�-d-glycopyranoside/Methyl-�-mannopyranoside (methyl-Man/Glc,Sigma Aldrich), �-d-mannose (Sigma Aldrich). Glycans used werebiotin-labeled polyacrylamide (PAA) conjugates or unlabeled PAAconjugates (Lectinity and American Consortium of Functional Gly-comics (www.functionalglycomics.org)), except for mannose-BSA(Sigma Aldrich), Mannotriose, 3′-sulfo-Lewisa, 3′-sulfo-LewisX,Blood group A and B (Dextra Labs), which were BSA-conjugatedand not biotinylated. Trichinella spiralis larvae were harvested frominfected mice and kindly provided by Dr. J. van der Giessen (RIVM,Bilthoven, the Netherlands), S. mansoni cercariae were kindly pro-vided by Dr. M. de Jong-Brink (FALW, VU University, Amsterdam,the Netherlands), and Haemonchus contortus larvae were a giftfrom Dr. J. Poot (Intervet Int., Boxmeer, the Netherlands). Solubleproducts (SP) of the helminth stages were prepared as described[36]. Larvae or cercariae were frozen in liquid nitrogen, and crushedwith mortar and pestle into powder. The powder was dissolvedin MilliQ and centrifuged to remove the insoluble fraction. After30 min of centrifugation at 10,000 × g at 4 ◦C, supernatants werefiltered (0.45 �m) and kept at −80 ◦C until further use. C. albicansyeast and hyphae were a kind gift from Prof. Dr. M. Netea (RUNMC,Nijmegen, the Netherlands). Different HIV proteins were providedby NIH AIDS Research and Reference Reagent Program, Divisionof AIDS, NIAID, NIH: HIV-1 SF162 gp140 trimer, catalog number:12026 from Dr. Leo Stamatatos; Recombinant HIV-1 IIIB gp120(CHO), catalog number: 11784 from DIAIDS, NIAID (produced byImmunodiagnostics); HIV-1 gp120 CM, catalog number: 2968from DAIDS, NIAID.
2.2. Cells
CHO, CHO Lec8 cells and Kato-III cells were cultured inRPMI1640 (Invitrogen) supplemented with 1000 U/ml peni-cillin/streptomycin (Lonza), 2 mM glutamine (Lonza) and 10%FBS (BioWhittaker). DCIR-Fc and DC-SIGN-Fc producing cell lineswere cultured in RPMI supplemented with 1000 U/ml peni-cillin/streptomycin, 2 mM glutamine, 60 �g/ml glutamic acid,60 �g/ml asparagine, 7 �g/ml adenosine, 7 �g/ml guanosine,7 �g/ml cytidine, 7 �g/ml uridine and 2.4 �g/ml thymidine (allfrom Sigma Aldrich), 1 mM MEM non-essential amino acids, 1 mMsodium pyruvate MEM (Invitrogen) and 10% dialyzed FBS (Invi-trogen). DCIR-Fc Lec8 cells were cultured in RPMI supplementedwith 1000 U/ml penicillin/streptomycin, 2 mM glutamine, 10% FBSand 500 �g/ml hygromycin (Invitrogen) to select for DCIR-Fc pro-ducing cells. Prostate cancer cell lines VCaP, PC346C, LNCaP andLAPC-4, were a kind gift of Dr. W.M. van Weerden (Erasmus MCRotterdam, the Netherlands). Colon cancer cell lines HCT116, HT-29 and SW480 were cultured in DMEM (Invitrogen) supplementedwith 1000 Units/ml penicillin/streptomycin mixture (Lonza) and10% FBS (BioWhittaker). Primary keratinocytes were obtained fromhealthy skin samples following plastic surgery, after obtaining
informed consent from all donors. Epidermis and dermis wereseparated as described previously [37]. The CD1a negative keratino-cytes were separated from the CD1a positive Langerhans cells usinganti-CD1a-labeled immunomagnetic microbeads (Miltenyi Biotec).
Ligands and glycans were coated on 96-well flat-bottomedLISA plates (Maxisorp, Nunc) overnight at room tempera-ure. Polyacrylamide (PAA)-glycans (5 �g/ml) and HIV-1 proteins10 �g/ml) were coated in 0.05 M Na2CO3 (pH 9.7). C. albicansyphae and yeast were coated in PBS (1 million particles/well). Sol-ble helminth products were coated at 10 �g/ml in 0.2 M NaHCO3pH 9.2). Plates were blocked with 1% BSA (Fraction V, Fatty acidree; PAA Laboratories) in TSM (20 mM Tris, pH 7.4, 150 mM NaCl,
mM CaCl2 and 2 mM MgCl2) buffer. DCIR-Fc (150 �g/ml) or DC-IGN-Fc (1.4 �g/ml) constructs were incubated in TSM for 3 h atoom temperature. After washing with TSM/0.05% Tween bindingas detected using 0.27 �g/ml goat-�-human-Fc PO in TSM/0.05%
ween for 30 min at room temperature. Binding was visualizedith 3,3′,5,5′-tetramethylbenzidine (TMB) as a substrate (Sigmaldrich) and optical density was measured by spectrophotometryt 450 nm. The calcium chelator EGTA (10 mM) was used to testhe calcium-dependency of the interaction. 50 mM of the monosac-haride �-d-mannose was used for competition inhibition. Controluman Fc binding was measured using 150 �g/ml human IgG1appa (Serotec).
.4. Flow cytometry
DCIR-, DC-SIGN- and control human-Fc coated beads were pre-ared as previously described [38]. DCIR-, DC-SIGN- and controluman-Fc coupled beads (40 beads/cell) were incubated with theifferent cell lines in TSM/0.05% BSA (Roche) for 45 min at 37 ◦C inhe presence or absence of 10 mM EGTA. Binding of DCIR- and DC-IGN and control human-Fc coupled beads was analyzed by flowytometry (FACScan, BD Biosciences). Expression of glycans wasvaluated using �-Lewis antibodies (5 �g/ml) and the lectin ConA10 �g/ml) in the presence of 25 mM and 100 mM Glc/Man block.
. Results
.1. DCIR interacts with sulfo-Lewisa, Lewisb, Lewisa andannotriose
Until recently, the glycan specificity of DCIR was debated, sinceinding to both mannose/fucose glycans and sulfated LacNAc andac, and biantennary N-glycans was observed [10,11]. We have con-rmed that fucose- and mannose-containing glycans act as DCIR
igands [12], similar to DC-SIGN. To compare the glycan specificityf DC-SIGN to that of DCIR in more detail, we tested the binding ofC-SIGN-Fc and DCIR-Fc to a panel of glycans (Supplementary Table). Binding of DC-SIGN was observed to mannose, mannotriose andll fucose-containing glycans tested, with the exception of sialy-ated Lewis structures and H-type 1 (Fig. 1a), confirming earlieresearch [17,39]. In the presence of EGTA binding of the glycansas inhibited to baseline levels demonstrating the specificity of
he Ca2+-dependent glycan binding. In contrast, DCIR binding couldot be observed to any of the tested glycans (Fig. 1b). This corre-ponds with our previous findings that glycosylated DCIR inhibitedts binding to immobilized glycans, due to occupation of the CRDf DCIR, since the only N-linked glycosylation site at position 185s located close to the glycan binding motif (position 195–197).o be able to investigate the glycan specificity of DCIR we madese of a DCIR-Fc construct produced in CHO Lec8 cells (DCIR-Fcec8), that synthesizes truncated complex N-glycans with terminal
-linked N-acetylglucosamine (GlcNAc) residues [40] due to the
ack of a functional UDP-Gal transporter [34]. DCIR-Fc produced inHO-Lec8 cells therefore contains truncated glycans, while DCIR-c produced in CHO cells is fully glycosylated. With the use of
tters 158 (2014) 33–41 35
DCIR-Fc Lec8 we could indeed reveal binding of DCIR to man-notriose, Lewisa, Lewisb and sulfo-Lewisa (Fig. 1c), which was,similar to DC-SIGN, blocked in the presence of EGTA, confirming theglycan specificity of DCIR for mannose- and fucose-containing gly-cans [12]. To demonstrate DC-SIGN- and DCIR-specific binding tothe DCIR-binding glycans Lewisb and sulfo-Lewisa, we investigatedthe DC-SIGN and DCIR binding in the presence of the monosac-charide mannose (Fig. 1d and e), since it is unknown whether theavailable DCIR antibodies act as blocking antibodies. Both DCIR andDC-SIGN binding to Lewisb and sulfo-Lewisa can be blocked by theaddition of the monosaccharide mannose, comparable to the EGTAcontrol. Since the DCIR binding strength in this assay is inferiorto that of DC-SIGN, the percentage of inhibition is lower, howeverthe signal after inhibition approaches the background signal. Thesedata demonstrate the specificity of both C-type lectins to interactwith the oligosaccharides in a Ca2+- and glycan-dependent manner.
Supplementary material related to this article can be found,in the online version, at http://dx.doi.org/10.1016/j.imlet.2013.11.007.
To compare the DCIR-glycan interactions with that of DC-SIGNto mannose- and fucose-containing glycan structures, differentconcentrations of neoglycoconjugates were immobilized and bind-ing of DC-SIGN-Fc and the different DCIR-Fc constructs was tested(Fig. 2). Sulfo-LewisX-BSA was included to evaluate the role of thesulfo-group in fucose-containing glycans. Since the composition ofglycoconjugates could influence glycan binding, we separated theglycans based on the carrier molecule. As expected, DC-SIGN inter-acted in a concentration dependent manner with coated glycans,which was already visible at the range of 0.2 to 1 �g/ml (Fig. 2). Incontrast, binding of DCIR-Fc was only detected at a concentrationof 10 �g/ml coated glycan, while binding of DCIR-Fc Lec8 could beobserved at somewhat lower concentrations. DCIR interacted moststrongly with sulfo-Lewisa and Lewisb and not with the fucose-containing glycan structures LewisX and LewisY. DC-SIGN bindingto sulfo-LewisX was negligible and the binding strength of DC-SIGNto sulfo-Lewisa was dependent on the glycoconjugate used for gly-can presentation, whereby DC-SIGN interacts more strongly withlow coating concentrations of sulfo-Lewisa-PAA (gray circle) com-pared to sulfo-Lewisa-BSA (black cross). In contrast, the bindingstrength of DCIR to sulfo-Lewisa-BSA was slightly increased com-pared to sulfo-Lewisa-PAA at a coating concentration of 5 �g/ml.
3.2. DCIR interacts with ligands of endogenous origin
DC-SIGN recognizes a wide variety of ligands which includepathogens as well as endogenous ligands, in both healthy and dis-eased state [6,18]. The overlapping glycan specificity of DCIR withthat of DC-SIGN suggests that DCIR may potentially interact withsimilar ligands as DC-SIGN. Since DCIR interacts most stronglywith sulfo-Lewisa and Lewisb, epithelial cells [41] and reportedchanges in sulfo-Lewisa expression during oncogenesis [42,43],may be recognized by DCIR. To investigate the binding of DCIRto potential sulfo-Lewisa- and/or Lewisb-expressing cells, we cou-pled DC-SIGN-Fc, the two DCIR-Fc constructs and a control humanIgG1 antibody to fluorescent beads and measured the binding ofthese beads to the different cells. The binding of CLR-Fc coupledbeads to cells is a well-established binding assay [38], in whicha high percentage of cells binding fluorescent beads indicates thepresence of cellular CLR-specific ligands. In Fig. 3a an example isgiven to illustrate how the calcium-dependent CLR-Fc binding iscalculated. We observed DCIR binding to keratinocytes, the gastriccancer cell line Kato-III and the colon carcinoma cell lines HCT116
and SW480, while binding was not observed to the colon carci-noma cell line HT-29 (Fig. 3b). DCIR-Fc coated on beads showedstrong binding, which was further increased when DCIR-Fc Lec8was used; indicating that clustering of DCIR on beads enhances the
36 K. Bloem et al. / Immunology Letters 158 (2014) 33–41
Fig. 1. DC-SIGN and DCIR binding to fucose- and mannose-containing glycans. Binding of DC-SIGN-Fc (A) and DCIR-Fc (B and C) to immobilized neoglycoconjugates.N SA, ma y of tD -BSA w
pbDrBsHTtcm((wcnL
eoglycoconjugates were attached to a PAA-tail, with the exception of mannose-B BSA-tail. The calcium chelator EGTA was used to test for the calcium-dependencC-SIGN-Fc and DCIR-Fc Lec8 to the DCIR-binding glycans Lewisb and sulfo-Lewisa
otency to interact with glycans exposed on cells compared to solu-le DCIR-Fc in the CLR binding ELISA. Since EGTA blocks DCIR- andC-SIGN-specific binding to a similar extent as the monosaccha-
ide mannose, only the Ca2+-dependent binding was investigated.inding of DC-SIGN to keratinocytes was not observed and oppo-ite of DCIR, low DC-SIGN binding was detected to HCT116, whileT-29 strongly interacted with DC-SIGN-Fc coated beads (Fig. 3b).o determine the expression of Lewis- and mannose-structures onhe different cells used, we stained the cells with Lewis-antigen spe-ific antibodies. In addition, the lectin conA, in combination withethyl �-d-mannopyranoside and methyl �-d-glucopyranoside
methyl-Man/Glc), was used to distinguish between diantennaryblocked with 25 mM methyl-Man/Glc) and oligomannose (blocked
ith 100 mM and higher concentrations of methyl-Man/Glc) gly-
an structures. Lewisa, Lewisb or oligomannose residues wereot detected on all cells tested (data not shown), while theewisa and Lewisb antibodies used in this study have been used
annotriose-BSA, Blood group A-BSA, Blood group B-BSA, which were attached tohe interaction. Values are represented as the mean + SD. (D and E) The binding ofas tested in the presence of EGTA and mannose block.
previously to demonstrate cellular expressed Lewisa and Lewisb
using the flow cytometer [44,45]. Unfortunately, we were unableto test the expression of sulfo-Lewisa on the cells, due to the lackof a commercially available antibody. Nevertheless, sulfo-Lewisa
expression has been reported for keratinocytes [41], gastric can-cer cells [42], HCT116 and SW480 [46], while this was absent onHT-29 cells [46], suggesting that the binding of DCIR to the firstmentioned cells is likely to be mediated by sulfo-Lewisa. High lev-els of LewisX and LewisY were present on HT-29 cells (data notshown), explaining the exclusive binding of DC-SIGN to these cells.
Prostate cancer cell lines potentially express Lewisb glycans[47]. The binding of DC-SIGN and DCIR to prostate cancer cell lineshas, to our knowledge, never been addressed before. Binding of
DCIR and DC-SIGN was observed to VCaP, PC346C and LNCaP cells,although the percentage of VCaP cells binding to DC-SIGN-Fc coatedbeads was substantially lower compared to the percentage of VCaPcells binding to DCIR-Fc coated beads (Fig. 4). DCIR and DC-SIGN
K. Bloem et al. / Immunology Letters 158 (2014) 33–41 37
F cocona jugate( A cont
bwc(s
TElbCoi
ig. 2. CLR-Fc binding is concentration-dependent. A concentration range of neoglynd DC-SIGN-Fc (E and F) was evaluated using the CLR binding assay. NeoglycoconB, D and F). Depicted is the Ca2+-dependent CLR-Fc binding by subtracting the EGT
inding to the prostate cancer cell lines could not be correlatedith the expression of Lewisb or Lewisa, since only a small per-
entage of the VCaP cells was positive for the expression of Lewisb
Table 1). A portion of the LNCaP cells was positive for the expres-ion of LewisX and LewisY, while PC346C and LAPC-4 cells did not
able 1xpression of Lewis-type and mannose-containing glycans on prostate cancer cellines. Glycan expression was determined with the use of Lewis antigen-specific anti-odies or with the mannose-specific lectin ConA in combination with Glc/Man block.ells were considered to express high-mannose structures when residual bindingf ConA was observed in the presence of Glc/Man block. Partial Lewis expressionndicates Lewis expression on less than 20 percent of the cells.
jugates was immobilized and binding of DCIR-Fc (A and B), DCIR-Fc Lec8 (C and D)s are separated based on the carrier molecule, being either PAA (A, C and E) or BSArol. One representative experiment out of 3 is shown.
express any Lewis-type glycans at all (Table 1). The partial expres-sion of Lewis structures on VCaP and LNCaP cells could not explainthe high percentage of cells binding DCIR-Fc and DC-SIGN-Fc coatedbeads. Nevertheless, the expression of oligomannose structureson the cells corresponded with the observed DCIR- and DC-SIGN-binding, suggesting that the interaction of DCIR and DC-SIGN withprostate cancer cell lines is likely mediated by mannose- instead offucose-containing glycans.
3.3. DCIR functions as pathogen recognition receptor
Apart from binding endogenous ligands the interaction of DC-SIGN with a wide variety of pathogens has been described [17],based on the presence of LewisX, LewisY, LDNF and high man-
nose structures. These include viruses, like HIV-1 [19], fungi,such as C. albicans [20] and helminths, like S. mansoni [24]. Toinvestigate whether DCIR also interacts with pathogens that bindDC-SIGN, we compared the binding activity of DC-SIGN and DCIR to
38 K. Bloem et al. / Immunology Letters 158 (2014) 33–41
F IGN-Fu -depeni the E
Htpcdmtaactgtg
Facr
ig. 3. DCIR and DC-SIGN binding to different cell types. Binding of DCIR-Fc and DC-Ssing flow cytometry. (A) The percentage of cells binding a fluorescent bead in a Ca2+
s extremely low. (B) Depicted is the Ca2+-dependent CLR-Fc binding by subtracting
IV-1 glycoproteins, C. albicans and the soluble products (SP) ofhe helminth species S. mansoni, T. spiralis and H. contortus. Likereviously reported, DC-SIGN was able to bind high mannose gly-an structures on both gp140 and gp120 HIV-1 proteins fromifferent strains (Fig. 5a) [19,48–50]. Since DCIR has affinity forannose-containing glycans, we investigated the DCIR interac-
ion with different strains, like the primary R5 strains SF162 [51]nd CM [52] and the laboratory-adapted X4 strain IIIB [51,53]. Inddition, since the HIV-1 envelop glycoprotein complex (Env) isomposed of three gp160 heterodimers, containing both gp120 andhe transmembrane glycoprotein gp41, we tested gp120 and gp140
lycoproteins. Gp140 HIV-1 proteins encompass the soluble part ofhe gp160 protein. Gp120 is heavily glycosylated, while the gp41lycosylation is less intense [50]. In sharp contrast with DC-SIGN,
ig. 4. DCIR and DC-SIGN binding to prostate cancer cell lines. Binding of DCIR-Fcnd DC-SIGN-Fc coated beads to different cancer cell lines was measured using flowytometry. Depicted is the CLR-specific binding with EGTA control subtracted. Oneepresentative experiment out of 2 is shown.
c coated beads to different cancer cell lines and primary keratinocytes was measureddent manner was calculated as shown in the histograms. The intra-assay variability
GTA control. One representative experiment out of 3 is shown.
DCIR-Fc showed no binding to HIV-1 proteins (Fig. 5b). Strikingly,DCIR-Fc Lec8 showed strong specific binding to the gp140 HIV-1glycoproteins, while the binding to gp120 was much lower. How-ever, in contrast to DC-SIGN, DCIR-Fc Lec8 did not interact withmannose-containing glycan structures present on the surface of C.albicans hyphae or yeast (Fig. 5c).
The interaction of DC-SIGN with soluble egg antigen (SEA) fromthe helminth S. mansoni depends on the expression of LewisX andthe helminth glycan LDNF [24]. Since DCIR did not interact withthese fucose-containing glycan structures it is expected not tointeract with soluble egg antigens (Fig. 1), however mannosylatedproteins are also frequently present on helminth species [54]. DC-SIGN-Fc bound cercariae SP of S. mansoni and larval SP of T. spiralis.Albeit lower, still significant binding of DCIR-Fc and DCIR-Fc Lec8 tothese helminth species was observed (Fig. 5). Although the bindingof DCIR-Fc Lec8 to larval SP of T. spiralis was slightly enhanced com-pared to DCIR-Fc, both proteins interacted equally with cercariaeSP of S. mansoni (Fig. 5). DCIR, as well as DC-SIGN, failed to interactwith larval SP of H. contortus.
In conclusion, we observed that DCIR binds a wide variety ofpathogens similar to DC-SIGN, although with a different specificity.While DCIR, similar to DC-SIGN, binds HIV-1, it prefers the gp140trimer over gp120 proteins. In contrast to DC-SIGN, DCIR did notinteract with C. albicans hyphae or yeast, while both lectins interactwith helminth products of S. mansoni and T. spiralis.
Our data indicate that, despite their overlapping glycan speci-ficities, DC-SIGN and DCIR recognize unique pathogenic andendogenous ligands, such as keratinocytes for DCIR and C. albicansfor DC-SIGN.
4. Discussion
In this study we compared the glycan specificity of DCIRwith that of DC-SIGN for neoglycoconjugates, pathogens and
K. Bloem et al. / Immunology Le
Fig. 5. DCIR and DC-SIGN binding to pathogens. (A–C) DCIR-Fc binding to knownDC-SIGN-binding pathogens was investigated using the CLR binding assay. RelativeFc binding compared to the non-coated control is shown. Human IgG1 was used tocontrol for background binding of the Fc portion to the HIV proteins and helminths.Is
eftrFgdctvDGtFwtesf�o
[19,31,59]. The DCIR interaction with gp140 HIV-1 proteins was
n addition, EGTA block was incorporated to control for CLR-specific binding. SP:oluble products.
ndogenous cell-associated ligands. Binding of DC-SIGN-Fc toucose-containing glycan structures was observed, with the excep-ion of H-type 1 and sialylated Lewis glycans, confirming earlieresearch [17,39]. We did not observe any glycan binding of DCIR-c, as we have demonstrated that the interaction of DCIR with itslycans is affected by glycosylation of the carbohydrate recognitionomain of DCIR [12]. The glycosylation of DCIR under physiologicalonditions can vary, depending on the glycosylation machinery ofhe cell and the presence of glycoside hydroxylases. In order to pre-ent the masking effect of the glycosylation of DCIR, we producedCIR-Fc in CHO Lec8 cells, that due to a lack of a functional UDP-al transporter [34] synthesize truncated complex N-glycans with
erminal �-linked GlcNAc residues [40]. We observed that DCIR-c Lec8 interacted predominantly with Lewisb and sulfo-Lewisa,hich is probably due to the presence of additional side groups in
hese glycan structures, which might contribute to an optimal ori-ntation of the �1-4 linked fucose. DC-SIGN has a broader glycanpecificity and binds �1-4, �1-2 and �1-3 linked fucose, there-
ore DC-SIGN is likely less dependent on the orientation of the1-4 linked fucose for its binding. In addition, the higher signalsbserved for DC-SIGN in the CLR binding assay can be explained by
tters 158 (2014) 33–41 39
the superior binding strength of DC-SIGN to glycans compared toDCIR.
CLR binding to endogenous ligands plays an important role inimmune homeostasis [18]. Furthermore, tumor cells can manipu-late DCs via CLR binding and thereby escape immune surveillance[55]. To identify endogenous cells expressing DCIR-binding gly-coproteins, various cancer cell lines and keratinocytes wereinvestigated for DCIR binding. The interaction of DCIR with pros-tatic cancer cell lines is probably mediated by mannosylatedproteins on the cell surface. These prostatic cancer cell lines bind themannose-binding lectin ConA in the presence of high concentra-tions methyl-Man/Glc, as shown in Table 1, suggesting the presenceof glycoproteins with high mannose glycan structures [56]. DCIRbinding to the gastric cancer cell line, Kato-III, the colon carci-noma cell lines, HCT116 and SW480, and the keratinocytes, mostlikely depends on the expected sulfo-Lewisa expression [41,42,46],since we could not detect the expression of Lewis-type glycansor high mannose glycan structures on these cells. Nevertheless,more research is needed to confirm that glycans involved in theobserved interaction contain either mannose or fucose residues.Testing CLR binding to cell lines in which specific glycosyltrans-ferases that are involved in the synthesis of sulfo-Lewisa or highmannose are knocked down, could give an indication for this. How-ever, it goes beyond the scope of this article to test if these glycansare indeed required for the observed interactions. The interactionof DC-SIGN with CEA on colorectal cancer cells is mediated by theexpression of LewisX and LewisY [28], however substantial LewisX
and LewisY is only present on HT-29, while all colorectal cancercell lines tested in this study have been described to express CEA[57,58]. Likely, DC-SIGN interacts with another glycoprotein andnot CEA on these cells. In addition, DCIR does not interact withLewisX and LewisY glycan structures (Fig. 1) and CEA is not presenton keratinocytes. Therefore newly unidentified glycoproteins arelikely to be involved in the binding of DCIR to cancer cells and kerati-nocytes. The identification of the glycoprotein ligands on the tumorcell lines, and the biological consequences of this interaction withDCIR need to be revealed in the future. Strikingly, keratinocytesexclusively bound DCIR, while DC-SIGN failed to interact with thesecells. Although the DC-SIGN-binding glycan sulfo-Lewisa is likelyexpressed on keratinocytes [41,42,46], the glycoconjugate carry-ing this glycan might not be optimal for DC-SIGN binding. On theother hand, other unidentified specific DCIR-binding glycans mightbe present on glycoproteins expressed by keratinocytes, mediatingthe exclusive DCIR-binding to keratinocytes.
We observed that DCIR binding to cellular expressed glyco-proteins is less dependent on the glycosylated state of DCIR asboth DCIR-Fc and DCIR-Fc Lec8 bind well to these endogenous lig-ands. This could be explained by the method used to test the CLRbinding to cellular expressed glycoproteins. Coupling of DCIR-Fcto fluorescent beads may form clusters and thereby enhance theavidity of the interaction. In addition, the glycan expression on thecell surface or on the glycoprotein scaffold is likely intense, whichmay contribute to an increased strength/avidity of the DCIR-glycaninteraction. Indeed, also the binding activity of DCIR to neogly-coconjugates is enhanced, when increasing the concentration ofcoated neoglycoconjugates (Fig. 2). Further research is needed fora detailed analysis on affinity interaction and avidity interaction ofDCIR with its ligands.
When we compared the recognition of pathogens by DCIRand DC-SIGN, we observed that DCIR, similar as DC-SIGN boundHIV-1 glycoproteins. This is in agreement with previous reportsshowing a role for both DCIR and DC-SIGN in HIV-1 infection
clearly enhanced over gp120 binding. Gp120 encompasses the sol-uble part of the HIV-1 envelope glycoprotein complex (Env). Env iscomposed of three gp160 heterodimers, containing both gp120 and
4 logy L
tgAictpworgbgia[Hi
oSaeyiSfilLbdbh
blcgptDb
C
A
dRLsPWc(cbCMwt
[
[
[
[
[
[
[
[
[
[
[
[
[
[
0 K. Bloem et al. / Immuno
he transmembrane glycoprotein gp41. Gp140 proteins comprise ap120 and a gp41 protein, which lack the transmembrane domain.lthough gp120 is heavily glycosylated and the gp41 glycosylation
s less intense [50], the glycan structures present on gp41 could stillontribute to the observed DCIR binding. Alternatively, the presen-ation of glycans on gp120 might differ between gp140 and gp120roteins. In vivo, DCs encounter complete HIV-1 particles coveredith Env, therefore recognition of gp140 appears to be more physi-
logically important in vivo, favoring a contribution of DCIR in HIV-1ecognition and pathogenesis [31,32]. In contrast, DCIR binding top120 HIV-1 proteins was relatively low. Slightly enhanced DCIRinding was observed for gp120 IIIB compared to gp120 CM. Thep120 IIIB is produced in CHO cells, which synthesize DCIR bind-ng glycans [12]. Nevertheless, gp120 CM is produced in insectsnd insect cells modify their N-glycans to oligomannose structures60], which are potential DCIR ligands as well. Alternatively, variousIV-1 strains might be differentially glycosylated, since variations
n DC-SIGN binding to different strains is also observed [48].The interaction of DC-SIGN with HIV-1 is based on the presence
f high-mannose structures [19]. This is also the case for the DC-IGN interaction with C. albicans [20]. We here show that DCIR hasffinity for mannose-containing glycans, similar to DC-SIGN, how-ver we could not observe binding of DCIR to C. albicans hyphae andeast. More research with different mannose-containing glycanss necessary to reveal differences in the affinity of DCIR and DC-IGN for varying mannose-glycans that can explain us why DCIRails to bind the mannosylated proteins present on C. albicans. Thenteraction of DC-SIGN with helminth species is, on the other hand,ikely mediated by the presence of the fucose-containing glycansewisX and LDNF [24,54]. These glycan structures are expressedy the helminth species S. mansoni and T. spiralis [61]. Since DCIRid not bind LewisX and LDNF, the DCIR-binding is likely mediatedy the expression of mannosylated proteins, frequently present onelminth species as well [54].
In conclusion, our report demonstrates for the first time DCIRinding to both endogenous as well as pathogenic glycosylated
igands. This binding is to a great extent influenced by the gly-osylation state of DCIR itself. Although DC-SIGN and DCIR sharelycan specificity, and share specificity for endogenous as well asathogenic ligands, our data demonstrate also different specifici-ies for these receptors. Future studies are necessary to explore howCIR- and DC-SIGN-binding ligands modify DC biology after ligandinding.
onflict of interests
The authors declare no conflicts of interest.
cknowledgments
We thank Dr. T. van Es for isolating the keratinocytes. Theifferent HIV proteins were provided by NIH AIDS Research andeference Reagent Program, Division of AIDS, NIAID, NIH. The CHOec8 cells were kindly provided by Prof. Dr. P. Stanley (Albert Ein-tein College Medicine, USA). The prostate cancer cell lines: VCaP,C346C, LNCaP and LAPC-4 were kindly provided by Dr. W. M. vaneerden (Erasmus MC Rotterdam, the Netherlands). The gastric
arcinoma cell line Kato-III was kindly provided by Dr. B. AppelmelkVU University Medical Center, the Netherlands) and the colon car-inoma cell lines: HCT116, HT29 and SW480 were kindly providedy Dr. R. Fijneman (VU University Medical Center, the Netherlands).
. albicans yeast and hyphae were kindly provided by Prof. Dr.. Netea (RUNMC, Nijmegen, the Netherlands). T. spiralis larvaeere kindly provided by Dr. J. van der Giessen (RIVM, Bilthoven,
he Netherlands), S. mansoni cercariae were kindly provided by
[
etters 158 (2014) 33–41
Dr. M. de Jong-Brink (FALW, VU University, the Netherlands), andH. contortus larvae were a gift from Dr. J. Poot (Intervet Int., theNetherlands). K. Bloem was supported by the Dutch Top InstitutePharma, project T1-214, I. M. Vuist was supported by MS Research(06-598), M. van den Berk was supported by GlycoPro EurostarsTI-124. J. J. García-Vallejo was supported by the Dutch AsthmaFoundation (3.2.10.040) and S. J. van Vliet was supported by a VENIgrant (863.10.017) from the Netherlands Organization for ScientificResearch (NWO).
References
[1] Matzinger P. The danger model: a renewed sense of self. Science2002;296:301–5.
[2] Medzhitov R, Preston-Hurlburt P, Janeway CAJ. A human homologue ofthe Drosophila Toll protein signals activation of adaptive immunity. Nature1997;388:394–7.
[3] Rock FL, Hardiman G, Timans JC, Kastelein RA, Bazan JF. A family of humanreceptors structurally related to Drosophila Toll. Proc Natl Acad Sci USA1998;95:588–93.
[4] Villadangos JA, Schnorrer P. Intrinsic and cooperative antigen-presentingfunctions of dendritic-cell subsets in vivo. Nat Rev Immunol 2007;7:543–55.
[5] Steinman RM, Banchereau J. Taking dendritic cells into medicine. Nature2007;449:419–26.
[6] Geijtenbeek TBH, van Vliet SJ, Engering A, ’t Hart BA, van Kooyk Y. Self- andnonself-recognition by C-type lectins on dendritic cells. Annu Rev Immunol2004;22:33–54.
[7] Sancho D, Reis e Sousa C. Signaling by myeloid C-type lectin receptors in immu-nity and homeostasis. Annu Rev Immunol 2012;30:491–529.
[8] Zelensky AN, Gready JE. The C-type lectin-like domain superfamily. FEBS J2005;272:6179–217.
[9] Bates EE, Fournier N, Garcia E, Valladeau J, Durand I, Pin JJ, et al. APCs expressDCIR, a novel C-type lectin surface receptor containing an immunoreceptortyrosine-based inhibitory motif. J Immunol 1999;163:1973–83.
10] Lee RT, Hsu T-L, Huang SK, Hsieh S-L, Wong C-H, Lee YC. Sur-vey of immune-related, mannose/fucose-binding C-type lectin receptorsreveals widely divergent sugar-binding specificities. Glycobiology 2011;21:512–20.
11] Hsu T-L, Cheng S-C, Yang W-B, Chin S-W, Chen B-H, Huang M-T, et al. Pro-filing carbohydrate–receptor interaction with recombinant innate immunityreceptor-Fc fusion proteins. J Biol Chem 2009;284:34479–89.
12] Bloem K, Vuist IM, van der Plas A-J, Knippels LMJ, Garssen J, García-Vallejo JJ,et al. Ligand binding and signaling of dendritic cell immunoreceptor (DCIR) ismodulated by the glycosylation of the carbohydrate recognition domain. PloSOne 2013;8:e66266.
13] Geijtenbeek TB, Torensma R, van Vliet SJ, van Duijnhoven GC, Adema GJ, vanKooyk Y, et al. Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3receptor that supports primary immune responses. Cell 2000;100:575–85.
14] Richard M, Veilleux P, Paquin R, Beaulieu D. The expression pattern of theITIM-bearing lectin CLECSF6 in neutrophils suggests a key role in the controlof inflammation. J Leukoc Biol 2002;71:871–80.
15] Eklöw C, Makrygiannakis D, Bäckdahl L, Padyukov L, Ulfgren A-K, LorentzenJC, et al. Cellular distribution of the C-type II lectin dendritic cell immunore-ceptor (DCIR) and its expression in the rheumatic joint: identification of asubpopulation of DCIR+ T cells. Ann Rheum Dis 2008;67:1742–9.
16] Lambert AA, Imbeault M, Gilbert C, Tremblay MJ. HIV-1 induces DCIR expressionin CD4+ T cells. PLoS Pathog 2010;6:e1001188.
17] Appelmelk BJ, van Die I, van Vliet SJ, Vandenbroucke-Grauls CM, Geijten-beek TBH, van Kooyk Y. Cutting Edge: carbohydrate profiling identifies newpathogens that interact with dendritic cell-specific ICAM-3-grabbing noninte-grin on dendritic cells. J Immunol 2003;170:1635–9.
18] García-Vallejo JJ, van Kooyk Y. Endogenous ligands for C-type lectin receptors:the true regulators of immune homeostasis. Immunol Rev 2009;230:22–37.
19] Geijtenbeek TB, Kwon DS, Torensma R, van Vliet SJ, van Duijnhoven GC, MiddelJ, et al. DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhancestrans-infection of T cells. Cell 2000;100:587–97.
20] Cambi A, Gijzen K, de Vries IJM, Torensma R, Adema GJ, Netea MG, et al. The C-type lectin DC-SIGN (CD209) is an antigen-uptake receptor for Candida albicanson dendritic cells. Eur J Immunol 2003;33:532–8.
21] Lozach P-Y, Lortat-Jacob H, de Lacroix de Lavalette A, Staropoli I, Foung S, AmaraA, et al. DC-SIGN and L-SIGN are high affinity binding receptors for hepatitis Cvirus glycoprotein E2. J Biol Chem 2003;278:20358–66.
22] Pöhlmann S, Zhang J, Baribaud F, Chen Z, Leslie GJ, Lin G, et al. Hepatitis C VirusGlycoproteins Interact with DC-SIGN and DC-SIGNR. J Virol 2003;77:4070–80.
23] Geijtenbeek TBH, van Vliet SJ, Koppel EA, Sanchez-Hernandez M,Vandenbroucke-Grauls CMJE, Appelmelk B, et al. Mycobacteria target
DC-SIGN to suppress dendritic cell function. J Exp Med 2003;197:7–17.
24] Van Die I, van Vliet SJ, Nyame AK, Cummings RD, Bank CMC, Appelmelk B, et al.The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosomamansoni egg antigens and recognizes the glycan antigen Lewis x. Glycobiology2003;13:471–8.
25] Geijtenbeek TBH, Krooshoop DJEB, Bleijs DA, van Vliet SJ, van Duijnhoven GCF,Grabovsky V, et al. DC-SIGN–ICAM-2 interaction mediates dendritic cell traf-ficking. Nature 2000;1:353–7.
26] Van Gisbergen KPJM, Ludwig IS, Geijtenbeek TBH, van Kooyk Y. Interactions ofDC-SIGN with Mac-1 and CEACAM1 regulate contact between dendritic cellsand neutrophils. FEBS Lett 2005;579:6159–68.
27] Van Gisbergen KPJM, Sanchez-Hernandez M, Geijtenbeek TBH, van KooykY. Neutrophils mediate immune modulation of dendritic cells throughglycosylation-dependent interactions between Mac-1 and DC-SIGN. J Exp Med2005;201:1281–92.
28] Van Gisbergen KPJM, Aarnoudse CA, Meijer GA, Geijtenbeek TBH, van KooykY. Dendritic cells recognize tumor-specific glycosylation of carcinoembryonicantigen on colorectal cancer cells through dendritic cell-specific intercellularadhesion molecule-3-grabbing nonintegrin. Cancer Res 2005;65:5935–44.
29] Saeland E, Belo AI, Mongera S, van Die I, Meijer GA, van Kooyk Y. Differentialglycosylation of MUC1 and CEACAM5 between normal mucosa and tumourtissue of colon cancer patients. Int J Cancer 2012;131:117–28.
30] Florentin J, Aouar B, Dental C, Thumann C, Firaguay G, Gondois-Rey F, et al.HCV glycoprotein E2 is a novel BDCA-2 ligand and acts as an inhibitor of IFNproduction by plasmacytoid dendritic cells. Blood 2012;120:4544–51.
31] Lambert AA, Gilbert C, Richard M, Beaulieu AD, Tremblay MJ. The C-typelectin surface receptor DCIR acts as a new attachment factor for HIV-1 indendritic cells and contributes to trans- and cis-infection pathways. Blood2008;112:1299–307.
32] Lambert AA, Barabé F, Gilbert C, Tremblay MJ. DCIR-mediated enhancementof HIV-1 infection requires the ITIM-associated signal transduction pathway.Blood 2011;117:6589–99.
33] Fawcett J, Holness C, Needham L, Turley H, Gatter K, Mason D, et al. Molecularcloning of ICAM-3, a third ligand for LFA-1, constitutively expressed on restingleukocytes. Nature 1992;360:481–4.
34] Deutscher SL, Hirschberg CB. Mechanism of Galactosylation in the Golgi Appa-ratus, A Chinese hamster ovary cell mutant deficient in translocation ofUDP-galactose across Golgi vesicle membrane. J Biol Chem 1986;261:96–100.
35] Geijtenbeek TBH, van Duijnhoven GCF, van Vliet SJ, Krieger E, Vriend G, FigdorCG, et al. Identification of different binding sites in the dendritic cell-specificreceptor DC-SIGN for intercellular adhesion molecule 3 and HIV-1. J Biol Chem2002;277:11314–20.
36] Klaver EJ, Kuijk LM, Laan LC, Kringel H, van Vliet SJ, Bouma G, et al. Trichurissuis-induced modulation of human dendritic cell function is glycan-mediated.Int J Parasitol 2013;43:191–200.
37] De Witte L, Nabatov A, Pion M, Fluitsma D, de Jong MAWP, de Gruijl T, et al.Langerin is a natural barrier to HIV-1 transmission by Langerhans cells. NatMed 2007;13:367–71.
38] Geijtenbeek TB, van Kooyk Y, van Vliet SJ, Renes MH, Raymakers RAP, Figdor CG.High frequency of adhesion defects in B-lineage acute lymphoblastic leukemia.Blood 1999;94:754–64.
39] Van Liempt E, Bank CMC, Mehta P, Garcia-Vallejo JJ, Kawar ZS, Geyer R, et al.Specificity of DC-SIGN for mannose- and fucose-containing glycans. FEBS Lett2006;580:6123–31.
41] Veerman EC, Bolscher JG, Appelmelk BJ, Bloemena E, van den Berg TK, NieuwAmerongen AV. A monoclonal antibody directed against high M(r) salivarymucins recognizes the SO3-3 Gal beta 1-3GlcNAc moiety of sulfo-Lewis(a): a
histochemical survey of human and rat tissue. Glycobiology 1997;7:37–43.
42] Zheng J, Bao WQ, Sheng WQ, Guo L, Le Zhang H, Wu LH, et al. Serum 3′-sulfo-Leaindication of gastric cancer metastasis. Clin Chim Acta 2009;405:119–26.
43] Yamori T, Ota DM, Cleary KR, Hoff S, Hager LG, Irimura T. Monoclonal antibodyagainst human colonic sulfomucin: immunochemical detection of its binding
[
tters 158 (2014) 33–41 41
sites in colonic mucosa, colorectal primary carcinoma, and metastases. CancerRes 1989;49:887–94.
44] Löfling J, Diswall M, Eriksson S, Borén T, Breimer ME, Holgersson J. Studies ofLewis antigens and H. pylori adhesion in CHO cell lines engineered to expressLewis b determinants. Glycobiology 2008;18:494–501.
45] Padró M, Mejías-Luque R, Cobler L, Garrido M, Pérez-Garay M, Puig S,et al. Regulation of glycosyltransferases and Lewis antigens expressionby IL-1� and IL-6 in human gastric cancer cells. Glycoconj J 2011;28:99–110.
46] Tsuiji H, Hayashi M, Wynn DM, Irimura T. Expression of mucin-associated sulfo-Lea carbohydrate epitopes on human colon carcinoma cells. Jpn J Cancer Res1998;89:1267–75.
47] Chandrasekaran EV, Chawda R, Locke RD, Piskorz CF, Matta KL. Biosynthesis ofthe carbohydrate antigenic determinants, Globo H, blood group H, and Lewisb: a role for prostate cancer cell alpha1,2-l-fucosyltransferase. Glycobiology2002;12:153–62.
48] Pöhlmann S, Baribaud F, Lee B, Leslie GJ, Sanchez MD, Hiebenthal-Millow K,et al. DC-SIGN interactions with human immunodeficiency virus Type 1 and 2and simian immunodeficiency virus. J Virol 2001;75:4664–72.
49] Kwon DS, Gregorio G, Bitton N, Hendrickson WA, Littman DR. DC-SIGN-mediated internalization of HIV is required for trans-enhancement of T cellinfection. Immunity 2002;16:135–44.
50] Eggink D, Melchers M, Wuhrer M, van Montfort T, Dey AK, Naaijkens BA, et al.Lack of complex N-glycans on HIV-1 envelope glycoproteins preserves proteinconformation and entry function. Virology 2010;401:236–47.
51] Stamatatos L, Cheng-mayer C. An envelope modification that renders a pri-mary, neutralization resistant clade B human immunodeficiency virus type 1isolate highly suspectible to neutralization by sera from other clades. J Virol1998;72:7840–5.
52] VanCott TC, Mascola JR, Loomis-Price LD, Sinangil F, Zitomersky N, McNeil J,et al. Cross-subtype neutralizing antibodies induced in baboons by a subtypeE gp120 immunogen based on an R5 primary human immunodeficiency virustype 1 envelope. J Virol 1999;73:4640–50.
53] Smith AL, Ganesh L, Leung K, Jongstra-Bilen J, Jongstra J, Nabel GJ. Leukocyte-specific protein 1 interacts with DC-SIGN and mediates transport of HIV to theproteasome in dendritic cells. J Exp Med 2007;204:421–30.
54] Van Die I, Cummings RD. Glycans modulate immune responses in helminthinfections and allergy. Chem Immunol Allergy 2006;90:91–112.
55] Aarnoudse CA, Garcia Vallejo JJ, Saeland E, van Kooyk Y. Recognition oftumor glycans by antigen-presenting cells. Curr Opin Immunol 2006;18:105–11.
56] Aarnoudse CA, Bax M, Sánchez-Hernández M, García-Vallejo JJ, van KooykY. Glycan modification of the tumor antigen gp100 targets DC-SIGN toenhance dendritic cell induced antigen presentation to T cells. Int J Cancer2008;122:839–46.
57] Herberman R, Aoki T, Cannon G, Liu M, Sturm M. Location by immunoelectronmicroscopy of carcinoembryonic antigen on cultured adenocarcinoma cells. JNatl Cancer Inst 1975;55:797–9.
58] Chakrabarty S, Chen JW, Chen XY, Trujillo JM, Lin PF. Modulation ofdifferentiation-related responses in human colon carcinoma cells by proteinkinase inhibitor H-7. Anticancer Res 1992;12:97–104.
59] Lee B, Leslie G, Soilleux E, Doherty UO, Baik S, Levroney E, et al. cis Expres-sion of DC-SIGN allows for more efficient entry of human and simianimmunodeficiency viruses via CD4 and a coreceptor. J Virol 2001;75:12028–38.
60] Tomiya N, Narang S, Lee YC, Betenbaugh MJ. Comparing N-glycan processing in
mammalian cell lines to native and engineered lepidopteran insect cell lines.Glycoconj J 2004;21:343–60.
61] Wisnewski N, McNeil M, Grieve RB, Wassom DL. Characterization of novelfucosyl- and tyvelosyl-containing glycoconjugates from Trichinella spiralismuscle stage larvae. Mol Biochem Parasitol 1993;61:25–35.
