A strategy for isolation of cDNAs encoding proteins affecting human intestinal epithelial cell growth and differentiation: characterization of a novel gut-specific N-myristoylated annexin.
The human intestinal epithelium is rapidly and perpetually renewed as the descendants of multipotent stem cells located in crypts undergo proliferation, differentiation, and eventual exfoliation during a very well organized migration along the crypt to villus axis. The mechanisms that establish and maintain this balance between proliferation and differentiation are largely unknown. We have utilized HT-29 cells, derived from a human colon adenocarcinoma, as a model system for identifying gene products that may regulate these processes. Proliferating HT-29 cells cultured in the absence of glucose (e.g., using inosine as the carbon source) have some of the characteristics of undifferentiated but committed crypt epithelial cells while postconfluent cells cultured in the absence of glucose resemble terminally differentiated enterocytes or goblet cells. A cDNA library, constructed from exponentially growing HT-29 cells maintained in inosine-containing media, was sequentially screened with a series of probes depleted of sequences encoding housekeeping functions and enriched for intestine-specific sequences that are expressed in proliferating committed, but not differentiated, epithelial cells. Of 100,000 recombinant phage surveyed, one was found whose cDNA was derived from an apparently gut-specific mRNA. It encodes a 316 residue, 35,463-D protein that is a new member of the annexin/lipocortin family. Other family members have been implicated in regulation of cellular growth and in signal transduction pathways. RNA blot and in situ hybridization studies indicate that the gene encoding this new annexin exhibits region-specific expression along both axes of the human gut: (a) highest levels of mRNA are present in the jejunum with marked and progressive reductions occurring distally; (b) its mRNA appears in crypt-associated epithelial cells and increases in concentration as they exit the crypt. Villus-associated epithelial cells continue to transcribe this gene during their differentiation/translocation up the villus. Immunocytochemical studies reveal that the intestine-specific annexin (ISA) is associated with the plasma membrane of undifferentiated, proliferating crypt epithelial cells as well as differentiated villus enterocytes. In polarized enterocytes, the highest concentrations of ISA are found at the apical compared to basolateral membrane. In vitro studies using an octapeptide derived from residues 2-9 of the primary translation product of ISA mRNA and purified myristoyl-CoA:protein N-myristoyltransferase suggested that it is N-myristoylated. In vivo labeling studies confirmed that myristate is covalently attached to ISA via a hydroxylamine resistant amide linkage. The restricted cellular expression and acylation of ISA distinguish it from other known annexins.(ABSTRACT TRUNCATED AT 400 WORDS)
Wice
B M
BM
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
Gordon
J I
JI
D000595:Amino Acid Sequence; D017302:Annexins; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D002454:Cell Differentiation; D002455:Cell Division; D003106:Colon / Q000502:physiology; D004247:DNA / Q000235:genetics; D004847:Epithelial Cells; D004848:Epithelium / Q000502:physiology; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000502:physiology*; D007583:Jejunum / Q000502:physiology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009226:Myristates; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D014407:Tumor Cells, Cultured
D017302:Annexins; D002135:Calcium-Binding Proteins; D009226:Myristates; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D004247:DNA