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Two beta-galactoside-binding proteins were found to be prominently expressed in the human colon adenocarcinoma T84 cell line. Cloning and sequencing of one, a 36-kDa protein, identified it as the human homolog of galectin-4, a protein containing two carbohydrate binding domains and previously found only in the epithelial cells of the rat and porcine alimentary tract. The other, a 29-kDa protein, is galectin-3, containing a single carbohydrate binding domain, previously found in a number of different cell types including human intestinal epithelium. Despite the marked similarities in the carbohydrate binding domains of these two galectins, their cellular distribution patterns are strikingly different and vary with cellular conditions. In confluent T84 cells, galectin-4 is mostly cytosolic and concentrated at the basal membrane, whereas galectin-3 tends to be concentrated in large granular inclusions mostly at the apical membrane. In subconfluent T84 cells, each galectin is distributed to specific domains of lamellipodia, with galectin-4 concentrated in the leading edge and galectin-3 more proximally. Such different localization of galectins-4 and -3 within T84 cells implies different targeting mechanisms, ligands, and functions. The localization of galectin-4 suggests a role in cell adhesion which is also supported by the ability of immobilized recombinant galectin-4 to stimulate adhesion of T84 cells.

L-29, a mammalian soluble lactose-binding lectin, was previously shown to be phosphorylated in confluent 3T3 fibroblasts (Cowles, E. A., Agrwal, N., Anderson, R. L., and Wang, J. L. (1990) J. Biol. Chem. 265, 17706-17712), which contain a small amount of this protein. We have determined the site of phosphorylation on L-29, taking advantage of the abundance of L-29 (about 1% of total soluble cell protein) in confluent polarized Madin-Darby canine kidney (MDCK) cells. Approximately 15-20% of the L-29 is phosphorylated in these cells. Phosphoamino acid analysis showed phosphate incorporation only at serine. Analysis of chymotryptic and endoproteinase Asp-N-generated NH2-terminal fragments by Edman degradation showed that 90% of the phosphate was at Ser6 and 10% at Ser12. The sequence surrounding Ser6, which is conserved in all known L-29 sequences, indicated that this serine might be phosphorylated by casein kinase I or casein kinase II. Reaction of human recombinant L-29 with [gamma-32P]ATP and each of these casein kinases showed that only casein kinase I catalyzed significant incorporation of 32P into L-29; and, as with the L-29 from the MDCK cell extracts, most of the phosphate was incorporated at Ser6 and a small amount was incorporated at Ser12.

A lactose-binding lectin from rat lung (RL-29) and a related lectin from Madin-Darby canine kidney (MDCK) cells have been analyzed with the primary goal of identifying post-translational modifications. The sequences show that RL-29 and the dog lectin are homologues of a lectin designated here as L-29 and elsewhere as CBP-35, epsilon BP, Mac-2, or L-34. RL-29 has a 140-amino-acid COOH-terminal carbohydrate-binding domain, a 20-amino-acid NH2-terminal domain, and an intervening domain consisting of 11 repeating elements rich in Pro, Gly, and Tyr (R-domain). The dog homologue has 14 repeating elements in its R-domain explaining its larger size. The sensitivity of the R-domain to bacterial collagenase allowed us to isolate the NH2-terminal domain and show that the NH2 terminus was blocked by acetylation and, in the accompanying paper (Huflejt, M. E., Turck, C. W., Lindstedt, R., Barondes, S. H., and Leffler, H. (1993) J. Biol. Chem. 268, 26712-26718), that the NH2-terminal domain is phosphorylated. In addition, we unexpectedly found an endogenous component, resembling 92-kDa type IV collagenase, that co-purified with L-29 and slowly digested the R-domain. Hence, L-29 is a substrate for bacterial and tissue collagenases even though the R-domain is non-collagenous. Moreover, the co-purification suggests a non-enzymatic interaction between 92-kDa collagenase and L-29.