A functional analysis of CD33 and CD34
In the bone marrow, CD34 is the best currently available marker of human multipotential stem cells and is downregulated upon the commitment of cells to the myeloid pathway. CD33 is the earliest marker of stem cell commitment to the myeloid lineage but is down-regulated as myeloid cells mature to granulocytes while expression is retained on monocytes, dendritic cells and macrophages. CD34 is a mucosialin and CD33 a member of the immunoglobulin superfamily. To determine the functions of CD34 and CD33 in early haematopoiesis, soluble forms of CD34 and CD33 have been constructed by PCR based construction of extracellular domain-IgGlFc (ECDFc) fusion plasmids. These chimaeric proteins have been used to define the ligand binding of CD33 and CD34 in a range of assay systems including: screening cell lines by immunofluorescence and iodination analysis; immunohistochemical staining; screening of cDNA libraries transiently expressed in COS cells by "panning" for ligands; and phosphorylation studies to assess the potential of phosphoproteins to regulate these molecules and their ligands in vitro. Iodination demonstrated that CD33 binds increasing numbers of ligands heterophilically on erythroleukemic (K562) and promonocytic (U937) cell lines at sizes ranging from 54-69Kd and 97-1 lOKd in a differentiation-dependent manner. CD33 was established to associate with several src-like kinases while Fc-CD33 precipitates phosphoproteins in the same region in KG-la, K562 and U937. Novel divalent-cation-dependent CD34 ligands of 66Kd and HOKd were isolated on HUVEC and both CD34 and Fc-CD34 precipitated phosphoproteins from HUVEC. From panning studies, Fc-CD33 demonstrated low levels of binding to ICAM-1 and a cDNA product sharing homology to the 3' end of dystrophin, termed here as apo-dystrophin-4. Apo-4 appears to give rise to two major proteins, at least one of which may provide an in vitro ligand for CD33 and contain a 3' enhancer. Models for both CD33 phosphoprotein and ligand binding behaviour are presented.