It has been hypothesized that human mucosal glucoamylase (EC 3.2.1.20 and 3.2.1.3) activity serves as an alternate pathway for starch digestion when luminal ␣-amylase activity is reduced because of immaturity or malnutrition and that maltase-glucoamylase plays a unique role in the digestion of malted dietary oligosaccharides used in food manufacturing. As a first step toward the testing of this hypothesis, we have cloned human small intestinal maltase-glucoamylase cDNA to permit study of the individual catalytic and binding sites for maltose and starch enzyme hydrolase activities in subsequent expression experiments. Human maltaseglucoamylase was purified by immunoisolation and partially sequenced. Maltase-glucoamylase cDNA was amplified from human intestinal RNA using degenerate and gene-specific primers with the reverse transcription-polymerase chain reaction. The 6,513-base pair cDNA contains an open reading frame that encodes a 1,857-amino acid protein (molecular mass 209,702 Da). Maltase-glucoamylase has two catalytic sites identical to those of sucrase-isomaltase, but the proteins are only 59% homologous. Both are members of glycosyl hydrolase family 31, which has a variety of substrate specificities. Our findings suggest that divergences in the carbohydrate binding sequences must determine the substrate specificities for the four different enzyme activities that share a conserved catalytic site.Starches are a mixture of two structurally different polysaccharides: amylose, a linear [4-O-␣-D-glucopyranosyl-D-glucose] n polymer, and amylopectin, with additional 6-O-␣-D-glucopyranosyl-D-glucose links (about 4% of total), which result in a branched configuration. Dietary starches are a mixture of approximately 25% amylose in amylopectin, a fact of nutritional significance because of the multienzyme complexity of the mammalian starch digestion pathway (1). ␣-amylase (EC 3.2.1.1) is the endoenzyme found in mature human salivary and pancreatic secretions that produces linear maltose oligosaccharides by hydrolysis of ␣134 linkages of amylose (2, 3). ␣-amylase bypasses the ␣1 3 6 linkages of amylopectin and produces branched isomaltose oligosaccharides. The starchderived oligosaccharides are not fermentable by yeast without further processing by ␤-amylase (EC 3.1.1.2), which hydrolyzes the nonreducing ends at 134 and 136 linkages (2). In mammals, hydrolysis of the nonreducing ends is carried out by small intestinal mucosal brush border-anchored sucrase-isomaltase (SIM) 1 (EC 3.2.1.48 and 3.2.1.10) and maltase-glucoamylase (MGA) (EC 3.2.1.20 and 3.2.1.3) complexes (1). Enzyme substrate specificities of SIM overlap with those of MGA. In vivo, SIM accounts for 80% of maltase (1,4-O-␣-D-glucanohydrolase) activity, all sucrase (D-glucopyranosyl-␤-D-fructohydrolase) activity, and almost all isomaltase (1, 6-O-␣-D-glucanohydrolase) activity (1). MGA accounts for all glucoamylase exoenzyme (1,4-O-␣-D-glucanohydrolase) activity for amylose and amylopectin substrates, 1% of isomaltase activity, and 20% of maltase activity (1...

N-Benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (PPH, human meprin), is a peptidase found in the microvillus membrane of human small intestinal epithelial cells. PPH belongs to the astacin family of zinc-metalloendopeptidases and is a protein complex composed of two glycosylated subunits, a and P.The present report describes the cloning of the complete /? subunit and the remaining N,-terminal end of the a subunit for analysis of their primary structures in addition to the examination of their biogenesis in transfected cell cultures. The complete open reading frame of the PPHP cDNA translates into 700 amino acid residues compared with 746 residues for the PPHa cDNA. The primary structure of P and a subunits are 44 % identical and 61 % similar. As predicted from their primary structure, the two subunits of PPH have identical modular structures; starting at the N,-terminus both contain a signal peptide, a propeptide, a protease domain containing the astacin signature, a meprin A5 protein tyrosine phospatase p (MAM) and a meprin and TRAF homology domain (MATH) domain, an epidermal growth factor(EGF)-like domain, a putative transmembrane anchor domain and a short cytosolic tail. Pulsekhase labelling and immuno-Gold electronmicroscopy of recombinant PPH , l ?and a subunits expressed in transfected MadinDarby canine kidney (MDCK) cells show that post-translational processing and transport of the two subunits are very different. When expressed alone, the subunit acquired complex glycan residues, readily formed homodimers and was transported to the plasma membrane. Small amounts of PPHP were found in the culture medium. In contrast, the cell-bound a subunit, when expressed alone, remained primarily in the high-mannose form, was aggregated and not expressed at the cell surface. However, the bulk of mostly endo-P-N-acetylglucosaminidase H-resistant a subunit was found in the filtered culture medium. The proteolytic event that leads to the formation of this soluble transport-competent form occurs in the endoplasmic reticulum (ER). Coexpression of the a subunit with the P subunit allowed the localisation of the a subunit to the plasma membrane. These studies indicate that assembly of the two subunits of PPH is required for the localisation of the a subunit to the plasma membrane. In contrast to rodent meprin, both PPH subunits are apically secreted from MDCK cells.Keywords: human meprin; N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase; astacin family; processing ; intracellular transport. N-Benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (PPH)is a metalloendopeptidase isolated from the microvillus membrane of human enterocytes [I]. The enzyme was first identified using N-benzoyl-L-tyrosyl-p-aminobenzoic acid (Bz-TyrNBzOH), a substrate used to assess exocrine pancreatic function Abbreviations. Bz-Tyr-NBzOH, N-benzoyl-L-tyrosyl-p-aminobenzoic acid; PPH, N-benzoyl-L-tyrosyl-I,-aminobenzoic acid hydrolase; EGF, epidermal growth factor; ER, endoplasmic reticulum; MAM, meprin A5 protein tyrosine phosphatase p ; MATH, meprin...

In this paper, we report the expression of PPH alpha in the polarized cell line MDCK (Madin Darby canine kidney). In these cells, the enzyme was synthesized in an inactive proform, which upon treatment with trypsin was activated. The enzyme isolated from cell extracts was core-glycosylated and appeared to be retained in the ER as a homodimer. No PPH alpha was detectable on the surface of intact cells by immunofluorescence. However, a complex glycosylated soluble but inactive form was present in the culture medium, suggesting that proteolytic removal of the C-terminal membrane anchoring peptide leads to the secretion of PPH alpha.

PABA peptide hydrotase (PPH) from human enterocytes is comprised of two subunits, alpha and beta. PPHa is over 70% identical to meprin, a protease isolated from mouse and rat kidney. The enzyme shows a modular organization in that it contains an astacin protease domain, an adhesive domain, an EGF-like domain, and a putative C-terminal membrane spanning domain. Expression of a chimeric meprin-PPHa cDNA in COS-1 cells led to the synthesis of immature, transport-incompetent homodimers. In addition, complex glycosylated forms were detected in the culture medium, suggesting that the enzyme is secreted after proteolytic removal of the membrane anchor.

No abstract