PMC

Colocalization profile of A33 antigen. Tight junction proteins are false-colored in red, and A33 antigen in green. a A33 partially colocalizes with the tight junction protein occludin. Immunofluorescence image of LIM1215 cells showing colocalization of A33 antigen and occludin. b A33 partially colocalizes with the actin-binding, tight junction-associated protein, ZO-1. Immunofluorescence image of LIM1215 cells showing colocalization of A33 antigen and ZO-1. c A33 partially colocalizes with the cytoskeleton. Immunofluorescence image of LIM1215 cells showing colocalization of A33 antigen and actin. Images have been false-colored for clarity

Overlays of A33 and five other CTLD structures in stick representations. (A) A33 (red) is overlaid with five other CTLDs (blue and yellow, at left) and is shown alone (at right and in panel B) to orient the reader. The other CTLDs are colored blue where they exhibit the more conserved portions of a C-type lectin-like fold and are colored yellow where they are not as conserved. Conserved secondary structure elements are labeled from N to C termini: β1, α1, α2, β2, β3, β4, and β5. Note that A33 does not contain a β4 strand. The β2 strand of A33 (red) and the β2 strands of the other CTLDs (“β2,” colored yellow for clarity) also superimpose closely. The orientation here is similar to the orientation of the A33 monomer on the right in Fig. 1A. CTLDs that have been found to be homodimers were chosen for comparison. (B) The view is rotated toward the viewer and to the left about 45° in order to see the “long loop.” Each of the five CTLDs has the “long loop” connecting the β2 and β3 strands, but A33 does not. Instead, A33 has a short connecting loop (residues 155 to 157). The legend to Fig. 3 identifies the five structures from which monomer CTLDs were extracted and superimposed on the A33 monomer.

The A33 dimer interface exhibits limited flexibility. The “B” monomers from each of the six A33 models were superimposed (red monomer at right). The relative positions of the “A” monomers at left (gray) are seen to vary among the six kinds of packing (see Table 2). The P2₁ crystal had two A33 dimers in the asymmetric unit. The A33 model from the “C2 (new)” crystal in which the A33 structure was determined is colored red.

Distribution of antibody-bound A33 antigen. a Cultured colon tumor epithelial cells contain little intracellular A33 antigen. Each of four cell lines were grown on coverslips, fixed, permeabilized, and A33 antigen was labeled with the monoclonal antibody m100.310*Alexa 488. Internal pools of antigen were seen only in LIM1215 cells. Bar represents 5 µm. b Antibody-bound A33 antigen shows persistent surface localization in a flow cytometric assay of dynamic internalization kinetics. Suspended cells were A33-labeled at 0°C and then incubated at 37°C, allowing labeled antigen to be internalized. After shifting cultures to 37°C, aliquots were removed and labeled with anti-mouse PE and analyzed flow cytometry in order to determine the fraction of labeled A33 that remained surface accessible over time. The level of surface-accessible A33 did not fall significantly below the level of total A33, indicating little internalization of the antibody-bound antigen in all cell lines tested except LIM1215. The decrease in total A33 signal is likely due to dissociation of the primary antibody, as the antigen is not shed