Phosphorylated human lectin galectin-3: analysis of ligand binding by histochemical monitoring of normal/malignant squamous epithelia and by isothermal titration calorimetry.
The human lectin galectin-3 is a multifunctional effector with special functions in regulation of adhesion and apoptosis. Its unique trimodular organization includes the 12-residue N-terminal sequence, a substrate for protein kinase CK1-dependent phosphorylation. As a step towards elucidating its significance, we prepared phosphorylated galectin-3, labelled it and used it as a tool in histochemistry. We monitored normal and malignant squamous epithelia. Binding was suprabasal with obvious positive correlation to the degree of differentiation and negative correlation to proliferation. The staining pattern resembled that obtained with the unmodified lectin. Basal cell carcinomas were invariably negative. The epidermal positivity profile was akin to distribution of the desmosomal protein desmoglein, as also seen with keratinocytes in vitro. In all cases, binding was inhibitable by the presence of lactose, prompting further investigation of the activity of the lectin site by a sensitive biochemical method, i.e. isothermal titration calorimetry. The overall affinity and the individual enthalpic and entropic contributions were determined. No effect of phosphorylation was revealed. This strategic combination of histo- and biochemical techniques applied to an endogenous effector after its processing by a protein kinase thus enabled a detailed monitoring of the binding properties of the post-translationally modified lectin. It underscores the value of using endogenous lectins as a histochemical tool. The documented approach has merit for applications beyond lectinology.
Szabo
P
P
1st Faculty of Medicine, Institute of Anatomy, Charles University, Prague, Czech Republic.
Dam
T K
TK
Smetana
K
K
Dvoránková
B
B
Kübler
D
D
Brewer
C F
CF
Gabius
H-J
HJ
D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D004847:Epithelial Cells / Q000737:chemistry* / Q000166:cytology; D004848:Epithelium / Q000737:chemistry / Q000378:metabolism* / Q000473:pathology; D037502:Galectin 3 / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D018307:Neoplasms, Squamous Cell / Q000737:chemistry / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D013194:Staining and Labeling
D037502:Galectin 3