Identification of Cell Surface Straight Chain Poly-N-Acetyl-Lactosamine Bearing Protein Ligands for VH4-34–Encoded Natural IgM Antibodies

Abstract

B cell binding and cytotoxicity by human VH4-34–encoded Abs of the IgM isotype has been well documented. A VH4-34-IgM has recently shown a favorable early response in a phase 1 trial for treatment of B cell acute lymphoblastic leukemia. Although its B cell ligand has been identified as straight chain poly-N-acetyl-lactosamine (SC-PNAL), the carrier of the sugar moiety has not been identified. Using nanoelectrospray ionization mass spectrometry, we identify the metabolic activation related protein complex of CD147-CD98 as a major carrier of poly-N-acetyl-lactosamine (SC-PNAL) on human pre-B cell line Nalm-6. Previous studies have suggested CD45 as the SC-PNAL carrier for VH4-34–encoded IgG Abs. Because Nalm-6 is CD45 negative, human peripheral blood B lymphocytes and human B cell line, Reh, with high CD45 expression, were examined for SC-PNAL carrier proteins. Western blot analysis shows that the CD147-98 complex is indeed immunoprecipitated by VH4-34–encoded IgMs from human peripheral blood B lymphocytes and human B cell lines, Reh, OCI-Ly8, and Nalm-6. However, CD45 is immunoprecipitated only from peripheral B lymphocytes, but not from Reh despite the high expression of CD45. These results suggest that human B cells retain SC-PNAL on the CD147-98 complex, but modulate the sugar moiety on CD45. Because the carbohydrate moiety may act as a selecting Ag for VH4-34 autoantibody repertoire, its differential expression on proteins may provide a clue to the intricate atypical regulation of the VH4-34 gene.

Innate-like B cell subsets during immune responses: Beyond antibody production

“…In contrast to what is observed under homeostatic conditions, circulating V H 4‐34 Abs are relatively easy to detect in some particular clinical conditions such autoimmune disorders including SLE and also different infectious diseases like mononucleosis, AIDS, or hepatitis C infection . In the context of self‐reactivity, it is known that these Abs recognize a conserved carbohydrate epitope, constituted by a linear chain of poly‐ N ‐acetyl‐lactosamine or i‐antigen present on human erythrocytes and B lymphocytes …”

“…37,138 One of the targets of trans-sialidase is CD45 that, once glycosylated, seems to trigger its phosphatase activity leading to activation of Src and BTK kinases in B cells; as this could easily explain the stimulation of any B cell, the classical downstream targets of BTK, including PKC-and NF-B, are not necessary for IL-17 production, 37 thus suggesting that a different, not yet characterized and maybe novel mechanism could be involved in this process. 143 In the context of self-reactivity, it is known that these Abs recognize a conserved carbohydrate epitope, constituted by a linear chain of poly-N-acetyl-lactosamine or i-antigen present on human erythrocytes and B lymphocytes. 144 Although there are many details that we still do not understand about this specific BCR-rearrangement and its implications in B cell functions beyond autoimmunity, some theories have been developed.…”

Clonal redemption and clonal anergy as mechanisms to balance B cell tolerance and immunity

“…A similar situation occurs in the 5%-10% of naive B cells within the normal human repertoire that express cell surface antibody employing the IGHV4-34 heavy chain element, which confers potentially pathogenic autoantibody activity by binding to mucin-like poly-N-acetyl-lactosamine self-glycans comprising the I/i blood antigen on the membrane of erythrocytes and on CD147 and CD45 on the surface of B cells. 41,42 This autoreactivity occurs regardless of what light chain IGHV4-34 heavy chains are paired with, and depends on a hydrophobic patch in IGHV4-34 framework region 1 that is recognized by anti-idiotype antibody 9G4. More specifically, a core tryptophan residue in the initial beta-strand (W7) in combination with a stretch of hydrophobic residues centered on positions 23-25 in the second beta-strand of the Ig fold has been shown to mediate interactions.…”

Ovarian carcinoma glyco-antigen targeted by human IgM antibody

“…Many mAbs derived from the VH4-34 heavy chain gene bind to a distinct straight chain poly n-acetyl lactosamine, referred to as the i/I antigen [ 2 , 3 ] and found on normal fetal red blood cells (RBCs) and mature B cells [ 4 ]. On glycan array (Consortium for Functional Glycomics), these VH4-34 encoded antibodies bind a long straight chain, poly N-acetyllactosamine with or without a terminal sialic acid [ 5 ]. Recently by using immuno-precipitation and Mass Spectrometry (Nano ESI), our group further identified the protein carrier of straight chain poly n-acetyl lactosamine epitope on B cell (including normal human B cells and human B cell lines) membrane as CD147/CD98 complex.…”

“…Recently by using immuno-precipitation and Mass Spectrometry (Nano ESI), our group further identified the protein carrier of straight chain poly n-acetyl lactosamine epitope on B cell (including normal human B cells and human B cell lines) membrane as CD147/CD98 complex. CD45 on human normal B cells also carry this carbohydrate structure [ 5 , 6 ].…”