Journal: The Journal of Biological Chemistry

Article Title: Engineering the fragment crystallizable (Fc) region of human IgG1 multimers and monomers to fine-tune interactions with sialic acid-dependent receptors

doi: 10.1074/jbc.M117.795047

Figure Lengend Snippet: Binding of IgG1-Fc variants to glycan receptors. A , mutants lacking the Asn-297 glycan are severely restricted in their capacity to bind DC-SIGN by ELISA. The addition of an N -linked sugar at position 221 results in proteins with a reduced capacity to bind DC-SIGN compared with their equivalent variants in which Asn-221 is absent. B , the hypersialylated D221N mutants bind Siglec-1. No binding was observed with the N297A/N563A glycan-deficient mutant ( error bars represent standard deviations around the mean value, n = 2 independent experiments).

Article Snippet: The same ELISA protocol used to detect DC-SIGN binding was used for human Siglec-1, Siglec-4, and Siglec-3 (Sino Biologicals).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Engineering the fragment crystallizable (Fc) region of human IgG1 multimers and monomers to fine-tune interactions with sialic acid-dependent receptors

doi: 10.1074/jbc.M117.795047

Figure Lengend Snippet: Model showing the contribution of different N -linked glycan and cysteine residues on Fc stoichiometry. The presence of Cys-575 allows optimal disulfide bonding between tail pieces of monomeric-Fcs. The tail piece glycan Asn-563 controls the number of monomeric tails that fit into the central corona (five to six in the case of hexa-Fc) while still allowing Cys-309 interdisulfide bridge formation. Cys-575 allows disulfide bonding between tail pieces of different monomers, but the absence of the Asn-563 glycan (the N563A mutant) allows many more tail pieces (up to twelve in the case of dodecamers) to fit into the central corona while still allowing disulfide bond formation through Cys-309 and/or Cys-575. The absence of Cys-575 prevents disulfide bonding between tail pieces, thereby generating sialylated monomers at Asn-563. The additional Asn-563 tail piece glycan in these monomers must explain the increased binding seen to Siglec-1 ( , A and B , and inset in this figure). The bulkier Asn-563 glycan with its predicted overall negative charge may lead to repulsion between two monomers, thus preventing disulfide bond formation between two Cys-309 residues in each monomeric Fc. The loss of both Asn-563 and Cys-575 (the N563A/C575A mutant) means that the observed laddered multimers must arise through Cys-309–mediated disulfide bonding in the Cγ2 domain. The presence of monomers, dimers, trimers, tetramers, pentamers, hexamers, and other intermediates in this mutant ( C ) suggests that these structures arise through a different mechanism, most likely via the sequential addition of 25-kDa half-mer Fc units at Cys-309. The lack of observable ladders with the L448STOP mutant implies that other amino acids in the tail piece are involved in bringing about monomer interactions that then facilitate disulfide bonding through either Cys-309 and/or Cys-575. Monomers with glycans located at both the N- and C-terminal ends of the Fc (Asn-221 and Asn-563) may allow for binding to receptors in cis as shown ( inset ).

Article Snippet: The same ELISA protocol used to detect DC-SIGN binding was used for human Siglec-1, Siglec-4, and Siglec-3 (Sino Biologicals).

Techniques: Mutagenesis, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Engineering the fragment crystallizable (Fc) region of human IgG1 multimers and monomers to fine-tune interactions with sialic acid-dependent receptors

doi: 10.1074/jbc.M117.795047

Figure Lengend Snippet: Binding of monomeric IgG1-Fc glycan variants to sialic acid-binding immunoglobulin-type lectins (Siglecs) with specificity for α2,3-linked sialic acid. A , the C575A monomer binds Siglec-1. B , the D221N/C575A monomer binds Siglec-1 and Siglec-4. ELISA as described under “Experimental procedures” with receptors coated down at 2 μg/ml and Fc-fragments at 20 μg/ml in TMS buffer ( error bars represent standard deviations around the mean value, n = 2 independent experiments).

Article Snippet: The same ELISA protocol used to detect DC-SIGN binding was used for human Siglec-1, Siglec-4, and Siglec-3 (Sino Biologicals).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell

Article Title: Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment

doi: 10.1016/j.cell.2020.08.001

Figure Lengend Snippet:

Article Snippet: Siglec 8 164Dy (7C9) , Fluidigm , Cat# 3164017B.

Techniques: Purification, Produced, Recombinant, Staining, Red Blood Cell Lysis, Saline, Blocking Assay, Labeling, Selection, Multiplexing, Amplification, Isolation, Software, Derivative Assay