Abstract
The myeloid C-type lectin dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN, CD209) recognizes oligosaccharide ligands on clinically relevant pathogens (HIV, Mycobacterium, and Aspergillus). Alternative splicing and genomic polymorphism generate DC-SIGN mRNA variants, which have been detected at sites of pathogen entrance and transmission. We present evidence that DC-SIGN neck variants are expressed on dendritic and myeloid cells at the RNA and protein levels. Structural analysis revealed that multimerization of DC-SIGN within a cellular context depends on the lectin domain and the number and arrangement of the repeats within the neck region, whose glycosylation negatively affects oligomer formation. Naturally occurring DC-SIGN neck variants differ in multimerization competence in the cell membrane, exhibit altered sugar binding ability, and retain pathogen-interacting capacity, implying that pathogen-induced cluster formation predominates over the basal multimerization capability. Analysis of DC-SIGN neck polymorphisms indicated that the number of allelic variants is higher than previously thought and that multimerization of the prototypic molecule is modulated in the presence of allelic variants with a different neck structure. Our results demonstrate that the presence of allelic variants or a high level of expression of neck domain splicing isoforms might influence the presence and stability of DC-SIGN multimers on the cell surface, thus providing a molecular explanation for the correlation between DC-SIGN polymorphisms and altered susceptibility to HIV-1 and other pathogens.
Highlights
Dendritic cells (DCs)-SIGN contains a carbohydrate-recognition domain, a neck region composed of eight 23-residue repeats, and a transmembrane region followed by a cytoplasmic tail containing recycling and internalization motifs (5, 30 –32)
Range of DC-SIGN Alternatively Spliced Isoforms—monocyte-derived dendritic cells (MDDCs) express a high number of alternatively spliced DC-SIGN mRNA species [36], which are found at mucosal HIV transmission sites [39]
Sequencing of the amplified fragments resulted in the identification of DC-SIGN mRNA species encoding for variants with alternative cytoplasmic tails and potentially soluble isoforms, with each group including transcripts differing in the neck domain or the carbohydrate-binding region (Fig. 1B)
Summary
Human peripheral blood mononuclear cells were isolated from buffy coats from healthy donors over a Lymphoprep (Nycomed, Norway) gradient according to standard procedures. PCR-generated fragments were resolved in agarose gels, purified, sequenced, and cloned into pCDNA3.1(Ϫ) vector. After 35 cycles of denaturation (95 °C, for 45 s), annealing (61 °C, for 30 s), and extension (72 °C, for 90 s), followed by a 10-min extension step at 72 °C, PCR-generated fragments were resolved in agarose gels, purified, cloned into pCR4-TOPO vector (Invitrogen), and sequenced. An expression vector for N-terminal-His epitope-containing DC-SIGN 1A (pCDNA3.1-DC-SIGN 1A-His) was created by PCR on pCDNA3.1-DC-SIGN 1A using oligonucleotides. COS-7 or HEK293T cells were transfected with SuperFect (Qiagen) using pCDNA3.1-based expression plasmids containing the distinct isoforms or mutants of the DC-SIGN cDNA. To generate DC-SIGN expression vectors lacking the lectin domain, PCR was performed on the pCDNA3.1-DC-SIGN 1A using oligonucleotides CD209s and CD209⌬Lectin (5Ј-CCCCAAGCTTGTCACAGGCGTTCCACTGCAGC-3Ј). Fragments containing either the full-length (8d⌬L) and a 7- and 6-repeat neck regions (repeats 1 through 7, 7d⌬L; repeats 1 through 6, 6d⌬L) were cloned into pCDNA3.1(Ϫ) to yield pCDNA3.1-DC-SIGN 8d⌬L, pCDNA3.1-DC-SIGN 7d⌬L, and pCDNA3.1-DC-SIGN 6d⌬L plasmids
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