MOLECULAR STRUCTURE OF ENDOSOMES IN DEVELOPING INTESTINE

During development, the absorptive cells of the intestine go through a
stage in which they are highly specialized for the endocytosis of
macromolecules from the intestinal lumen. During this time (which,
depending upon the species, may occur in utero or after birth) these
cells contain an elaborate endosomal complex. This complex has been
suggested to be important in the selective transepithelial transport of
growth factors that are crucial for the normal development of the
intestine and other organ systems. However, little is known about the
cellular mechanisms that control the selective transepithelial transport
of macromolecules while preventing pathogens from opportunistically
infecting through the same route. To identify the mechanisms that
underlie selective transepithelial transport, it is necessary to
characterize the compartment involved in sorting of internalized
macromolecules. The goals of the current proposal are to obtain the cDNA
sequence for an integral membrane glycoprotein (entubin), which is found
in the endosomes of the developing intestine of several species,
including human, but is absent from the mature intestine or any other
cell type. A rat neonatal intestine cDNA library will be screened by
hybridization using oligonucleotide probes generated after obtaining
internal amino acid sequence from affinity purified entubin. In the rat,
entubin is present in two electrophoretically distinguishable forms,
indicating either alternative splicing of mRNAs, multiple genes, or
differential glycosylation. Therefore, this cDNA will be used to
characterize the number and size of transcriptional units encoding
entubin and to determine the number of genes encoding entubin. In
addition, the cDNA will be used to screen other epithelial cell and non-
epithelial cell types for entubin-related proteins. If entubin related
proteins are detected in other cell types, it is possible that entubin
(or entubin-like proteins) are required for the normal function of
endosomes. In addition, the carbohydrate composition of this
glycoprotein will be determined by specific glycosidase digestion of
affinity purified entubin and together with the deduced amino acid
sequence used to predict the topology of the protein in the membrane.
This characterization of entubin is a first step in understanding the
molecular structure of endosomal membranes. Also, although the function
of entubin is unknown, it provides a unique and valuable marker of the
endosomal compartment. By transfecting cell lines with the cDNA for
entubin it will be possible to carry out detailed morphological and
biochemical studies of the targeting of endosomal proteins and to address
the questions of biogenesis of endosomal compartments, endosomal
dynamics, and recycling of endosomal components. In the long term, these
studies will provide insight into the mechanism of sorting and targeting
of developmentally important ligands such as growth factors.