DEVELOPMENT AND CONTROL OF SYNTHESIS AND SECRETION

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The lactate-phlorizin hydrolase (LPH) gene encodes a disaccharidase crucial for the digestion and absorption of lactose, the main carbohydrate in milk, and thus plays a critical role in the nutrition of the mammalian neonate. Except for transient expression in the fetal colon, LPH is found only in the absorptive cells of the small intestine. Among the well characterized microvillus membrane enzymes, LPH is unique in its pattern of expression and its developmental regulation. Over the past five years of this program, essential features of LPH structure and expression have been delineated in both animals and humans. It is now clear that: 1) all substrate specificities of the active site in the rat are found at a single locus; 2) LPH expression begins concurrently with morphogenesis of fetal rat small intestinal epithelium; 3) the cryptvillus (vertical), proximal to distal (horizontal), and chronologic (developmental) axes, as well as human genetic/racial lactase levels are primarily regulated at the level of gene transcription; and 4) LPH mRNA is targeted to the apical pole of the enterocyte in both animals and humans. The central hypothesis underlying the studies proposed in the present application is that unique factors regulate the spatial and chronologic expression of this important protein and that understanding essential processes of regulation of human LPH will yield insights into enterocyte gene expression in general. Accordingly, an integrated series of experiments has been formulated to delineate the molecular basis of LPH function, using human material and, when indicated, animals. We shall first define the structural requirements of the human LPH active site, and define the domains involved in LPH processing. Next, we shall define mechanism(s) of regulation of human LPH gene expression. The 5'-flanking region of human LPH will be cloned and characterized, and its functional domains defined using transient transfection assays and transgenic mice. We shall identify and clone genes for trans-acting factors which confer cell-specific and age-dependent genetic/racial regulation of human LPH expression. We shall elucidate the role of mRNA targeting in LPH expression by identifying the sequences in the 3'-untranslated region of LPH mRNA which bind potential targeting proteins and confirm the functional significance of sequences in the 3'-UTR responsible for apical localization. Having established that the fundamental regulation of LPH is transcriptional, and having the conceptual framework, techniques and reagents necessary to define structure/function relationships in this unique gene, we are poised to elucidate detailed mechanisms of human LPH gene regulation.