Epitope recognition of antibodies that define the sialomucin, endolyn (CD164), a negative regulator of haematopoiesis

Abstract

Endolyn (CD164) is a sialomucin that functions as an adhesion molecule and a negative regulator of CD34+ CD38- human haematopoietic precursor cell proliferation. The 105A5 and 103B2/9E10 CD164 monoclonal antibodies (mAbs), which act as surrogate ligands, recognize distinct glycosylation-dependent classes I and II epitopes located on domain I of the native and recombinant CD164 proteins. Here, we document five new CD164 mAbs, the 96 series, that rely on conformational integrity, but not glycosylation, of exons 2- and 3-encoded CD164 domains, thereby resembling the class III mAbs, N6B6 and 67D2. Although all the 96 series class III mAbs labelled both the 105A5+ and 103B2/9E10+ cells, cross-competition and immunoblotting studies allow them to be categorized into two distinct class III subgroups, i.e. the N6B6-like subgroup that only recognizes 80-100 kDa proteins and the 67D2-like subgroup that also recognizes a higher molecular weight (>220 kDa) form. To more closely define the reactivity patterns of mAbs to the classes I and II epitopes, the global glycosylation patterns of the soluble human (h) CD164 proteins were determined using lectin binding, high-performance liquid chromatography (HPLC) and mass spectrometry. hCD164 recombinant proteins bound to the lectins, Galanthus nivalis agglutinin, Datura stramonium agglutinin, Sambucus nigra agglutinin, Maackia amurensis agglutinin and peanut agglutinin, indicating the presence of high mannose and complex N-glycans, in addition to core 1 O-glycans (the Tn antigen) and alpha2-3 and alpha2-6 sialic acid moieties. Our HPLC and mass spectrometry results revealed both high mannose and complex N-glycosylation with various numbers of branches increasing the complexity of the glycosylation pattern. Most O-glycans were small, core 1 or 2 based. High levels of sialylation in alpha2-3 and alpha2-6 linkages, without sialyl-Lewis X, indicate that the majority of these hCD164 recombinant proteins are unable to bind to selectins in our assay system, but may interact with Siglec molecules.

Endolyn (CD164) modulates the CXCL12-mediated migration of umbilical cord blood CD133+ cells

“…For example, this chemokine/receptor axis promotes the intramedullary migration of HSCs/HPCs by activation of the integrins very late antigen-4 (VLA-4) and 28,29 Here, we provide evidence that endolyn (CD164) is a key molecule regulating the CXCL12-mediated migration of human umbilical cord blood (UCB) CD133 ϩ cells, and we demonstrate that CD164 acts as a component of a CXCR4 complex. CD164 is a type 1 integral transmembrane sialomucin (for reviews, see Watt and Chan,30 [34][35][36] Class III mAbs recognize a ubiquitously expressed epitope on the CD164 peptide backbone. [34][35][36] Epitopes recognized by the CD164 mAbs are expressed by most CD133 ϩ HSCs/HPCs, albeit at varying levels, 34 with the class II epitope the most extensively studied.…”

“…10,28,29 Here, we provide evidence that endolyn (CD164) is a key molecule regulating the CXCL12-mediated migration of human umbilical cord blood (UCB) CD133 ϩ cells, and we demonstrate that CD164 acts as a component of a CXCR4 complex. CD164 is a type 1 integral transmembrane sialomucin (for reviews, see Watt and Chan, 30 Chan et al, 31 Zannettino et al, 32 Watt et al, 33 Watt et al, 34 Jorgensen-Tye et al, 35 Doyonnas et al, 36 and Buhring et al 37 ) containing 2 extracellular O-glycan-substituted mucin domains (I and II) linked by a cysteine-rich nonmucin domain. There are 3 classes of CD164 monoclonal antibodies (mAbs).…”

“…There are 3 classes of CD164 monoclonal antibodies (mAbs). [34][35][36] Class I and II mAbs identify glycosylated variants/epitopes of CD164 that have restricted patterns of cell expression. [34][35][36] Class III mAbs recognize a ubiquitously expressed epitope on the CD164 peptide backbone.…”

“…[34][35][36] Class I and II mAbs identify glycosylated variants/epitopes of CD164 that have restricted patterns of cell expression. [34][35][36] Class III mAbs recognize a ubiquitously expressed epitope on the CD164 peptide backbone. [34][35][36] Epitopes recognized by the CD164 mAbs are expressed by most CD133 ϩ HSCs/HPCs, albeit at varying levels, 34 with the class II epitope the most extensively studied.…”

“…[34][35][36] Class III mAbs recognize a ubiquitously expressed epitope on the CD164 peptide backbone. [34][35][36] Epitopes recognized by the CD164 mAbs are expressed by most CD133 ϩ HSCs/HPCs, albeit at varying levels, 34 with the class II epitope the most extensively studied. Interestingly, CD164 class II epitope distribution on HSCs/HPCs and pre-B precursors from human bone marrow is reminiscent of functionally absent cells in CXCR4-and CXCL12-deficient mice, suggesting a potential association between the 2 receptors and bone marrow homing or retention.…”