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.

Abstract

Abstract. 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 HT29 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.

cDNA Isolation and Gene Expression of the Maize Annexins p33 and p35

“…Outside the plant kingdom, the maize annexins are most similar to annexin XHIa, originally described as an intestine-specific form of human annexin I (Wice and Gordon, 1992). The association of this annexin with the apical membrane of secretory cells of the intestinal epithelium suggests a role in the secretory pathway, a finding that is made more compelling by the recent discovery of annexin XHIb .…”

“…The most similar nonplant annexin is the "intestine-specific" form of human annexin I, now classed as annexin XHIa (35% identity; see Figs. 1 and 2) (Wice and Gordon, 1992;. Expression in Escherichia coli yields a 34-kD protein on SDS-PAGE, which strongly cross-reacts with antibodies to coleoptile p33/p35 (data not shown).…”

“…The longest cDNA clone was sequenced and its Comparison of annexin identities. The percentage identities were calculated pairwise for the amino acid sequences predicted from coding regions of full-length annexin cDNAs from maize (ZMANN33, ZMANN35), bell pepper (CAANN; Proust et al, 1996), and humans (HSANNXIII; Wice and Gordon, 1992), and partial cDNAs from alfalfa (MSANN; Pirck et al, 1994) and strawberry (FAANN; Wilkinson et al, 1995). The identities were calculated using the PALIGN program in PCGENE (Intelligenetics).…”

Immunolocalization of a novel annexin‐like protein encoded by a stress and abscisic acid responsive gene in alfalfa

“…A putative phosphorylation site for protein kinase C, as well as casein kinase II, can be detected near the N-terminus. Between positions 265 and 272 the TLIRVIVTR sequence highly resembles the TLIRIVVTR motif, the actin bundling site of annexin II, that is also present in human annexin XIIIa Wice and Gordon, 1992). Further experiments are required to test whether AnnMs2 can bind to actin as was shown for tomato annexins p34 and p35 (Calvert et al, 1996).…”

Annexins of Plant Cells

“…Peptide sequences obtained from tomato (Smallwood et al, 1990), corn (Blackbourn et al, 19921, pea (Clark et al, 19921, and cotton (Andrawis et al, 1993) proteins a11 indicated a degree of primary sequence conservation between plant and animal annexins. In animal cells most of the annexins are acetylated on their N terminus, although there is one report of an annexin with a myristolated N terminus (Wice and Gordon, 1992). Most of the plant proteins are also N-terminally blocked, but the nature of chemical modification is unknown.…”