Abstract
Expression and synthesis of sucrase-isomaltase (SI) were studied in human jejunum and in the colon tumor cell lines Caco-2 and HT-29. Twelve monoclonal antibodies produced against the adult human intestinal enzyme were shown to recognize specifically SI by immunoprecipitation of 14C-labeled membrane proteins, analysis of enzyme activities in the immunoprecipitates, and immunoblotting. These antibodies produced markedly different patterns of immunofluorescent staining of the intestinal mucosa. Three of them were specific for the absorptive villus cells, while the other nine also stained the luminal membrane of the proliferative crypt cells, with different intensities which paralleled their ability to recognize SI in immunoblots. Sequential immunoprecipitation of SI solubilized from purified brush borders or entire jejunum with four selected antibodies demonstrated the presence of different forms of the enzyme, expressed by either villus or crypt cells. Two immunologically distinct forms of high mannose precursor (hmP1 and hmP2) were also identified in both jejunal mucosa and colon tumor cells. They were present as monomers and their immunological differences were preserved under various ionic and pH conditions. Pulse-chase studies indicated that, in Caco-2 cells, hmP1 is converted into hmP2 within 30 min of chase, and hmP2 is then processed into the complex-glycosylated precursor destined for the brush border membrane. hmP1 was immunologically related to the mature SI present in crypt cells and lacked the epitopes specific for mature SI expressed by villus cells. These results demonstrated that sucrase-isomaltase is synthesized by both crypt and villus cells, but processing of the cotranslationally glycosylated high mannose precursor is dependent on the state of differentiation of the enterocytes. This may represent a general mechanism for the regulation of expression of differentiated cell products at the post-translational level.
Highlights
Expression and synthesis of sucrase-isomaltase (SI) The adult intestinal epithelium is a system of continuous were studied in human jejunumand in thceolon tumor cell renewal consisting of spatially separated stem cells, procell linesCaco-2 and HT-29
Sequential immunoprecipitation of SI solubilized from purified brush borderosr entirejejunum with four selected antibodies demonstrated the presence of different forms of the enzyme, expressed by either villus or crypt cells
It is generally assumed that thelower crypt cells do not synthesize most brush border enzymes typical of villus cells, and cell differentiation may be controlled primarily at thelevel of transcription (5,6)
Summary
Preparation of MonoclonalAntibodies HSI 1-13-Three independent fusions yielded approximately 650 hybridoma cultures, and 62of them produced antibodies reacting with formaldehyde-fixed frozen sections of human jejunum in the “Experimental Procedures” presented in miniprint at the end of this paper. Five of them appeared specific for onlyone of the two subunits of SI:HSI 3 (blot 2,4, HSI 9 (blot 4, J), and HSI 10 (blot, J) for sucrase ( S ) ;HSI 12 (blot 6,J)and HSI 14 (blot 7,4 for isomaltase (I) These five antibodies recognized the single chain precursor (cP)of SI present in membranes of both human jejunal brush borders and Caco-2 cells (blots ). To rule out the possibility that the different forms of SI cells with an irregular and weak staining of the apical mem- identifiedwith the sequential incubations described above brane; HSI 8 stained intensely the entire surface membrane were an artefact produced by [‘4C]formaldehyde labelingth, e andthe cytoplasm of upper-crypt to mid-villuscells (not same procedure of extraction/depletion presented in Fig. 4a shown). Sucrase-isomaltase-relatedpolypeptides are abbreviated as follows: cP, complex glycosylatedprecursor; hmP, high mannose precursor; S, sucrase subunit; I, isomaltase subunit
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