Topology and quaternary structure of pro-sucrase/isomaltase and final-form sucrase/isomaltase.

Pig sucrase/isomaltase (EC 3.2.1.48/10) was purified from intestinal microvillar vesicles prepared from animals with and without pancreatic-duct ligation to obtain the single-chain pro form and the proteolytically cleaved final form respectively. The purified enzymes were re-incorporated into phosphatidylcholine vesicles and analysed by electron microscopy after negative staining. The two forms of the enzyme were observed as identical series of characteristic projected views that could be unified in a single dimeric model, containing two sucrase and two isomaltase units. This shows a homodimeric functional organization similar to that of other microvillar hydrolases. The bulk of the dimer was separated from the membrane by a maximal gap of 3.5 nm, representing a junctional segment connecting the intramembrane section of the anchor to the catalytically active domain of sucrase/isomaltase. The enzyme complex protrudes from the membrane for a distance of up to 17 nm. From charge-shift immunoelectrophoresic studies of hydrophilic prosucrase/isomaltase and from electron microscopy of reconstituted pro-sucrase/isomaltase, there was no evidence to suggest the presence of anchoring sequences between the sucrase and isomaltase subunits.     

Feeding rats a high fat/carbohydrate ratio diet reduces jejunal S/I activity ratio and unsialylated galactose on glycosylated chain of S–I complex

AimsWe examined whether decreasing jejunal sucrase/isomaltase (S/I) activity ratio by feeding rats a high fat/carbohydrate ratio diet is regulated by changing glycosylated chains on the S–I complex.Main methodsJejunal activities of sucrase, isomaltase and β-1,4-galactosyltransferase were examined in rats fed a high fat/carbohydrate or a low fat/carbohydrate ratio diet. The amount of galactose and mannose in the glycosylated chain on the S–I complex in rats fed both diets was determined using RCA₁₂₀ and Con A lectins, respectively. The effects of reducing unsialylated galactose from the glycosylated chain on the S–I complex were assessed by determining sucrase activity in purified S–I complex treated with β-galactosidase.Key findings and significanceFeeding rats a high fat/carbohydrate ratio diet reduced jejunal S/I activity ratio in mucosal homogenates and purified fractions. The level of unsialylated galactose in glycosylated chains on the S–I complex was reduced by feeding rats a high fat/carbohydrate ratio diet. The form with reduced levels of unsialylated galactose had lower sucrase activity than that with more unsialylated galactose. The reduction of galactose on the S–I complex by β-galactosidase in vitro reduced sucrase activity. Feeding rats a high fat/carbohydrate ratio diet also reduced jejunal β-1,4-galactosyltransferase activity. Taken together, decreasing the S/I activity ratio by feeding rats a high fat/carbohydrate diet is associated with the reduction of unsialylated galactose on the glycosylated chain of the S–I complex.     

A two-active site one-polypeptide enzyme: the isomaltase from sea lion small intestinal brush-border membrane. Its possible phylogenetic relationship with sucrase-isomaltase

The enzyme responsible for all of the isomaltase activity and much of the maltase activity in the small intestine of the Californian sea lion (Zalophus californianus) was isolated by detergent solubilization of the brush-border membrane, followed by immunoadsorption chromatography using antibodies directed against rabbit sucrase-isomaltase. In 0.1% Triton X-100, sea lion isomaltase occurs as a monomer of Mr = 245,000 and is composed of a single polypeptide chain. As judged from the stoichiometry of the covalent binding of the affinity label, conduritol-B-epoxide, this polypeptide chain carries two enzymatically active sites; they are apparently identical and do not show either positive or negative cooperativity. In addition to cross-reacting immunologically with rabbit sucrase-isomaltase, sea lion isomaltase has similar overall enzymatic properties, with the exception of not hydrolyzing sucrose. The Alaskan fur seal (Collarhinus ursinus) has a two-active site isomaltase; however, in contrast to the sea lion, this animal is endowed with a small but significant sucrase activity. Along with (fully active) pro-sucrase-isomaltase, sea lion isomaltase is one of the very few examples of enzymes with more than one active site on a single polypeptide chain acting "in parallel" (rather than "in series"). Furthermore, this enzyme triggers some interesting questions on the phylogenetical pedigree of small intestinal sucrase-isomaltase.     

The mode of anchoring and precursor forms of sucrase-isomaltase and maltase-glucoamylase in chicken intestinal brush-border membrane. Phylogenetic implications

Chicken intestinal sucrase-isomaltase and maltase-glucoamylase have been isolated in their intact form by detergent solubilization and characterized as to their subunit composition and mode of anchoring in the brush-border membrane. Both are heterodimeric enzyme complexes composed of two subunits each of approximately 140 and 130 kDa. Contrary to the mammalian sucrase-isomaltase, chicken isomaltase was identified as the smaller of the two subunits. As was shown by hydrophobic labeling, only one of the two subunits in each heterodimer is anchored in the bilayer, the smaller 130 kDa isomaltase subunit of the sucrase-isomaltase complex, and the larger 140 kDa subunit of the maltase-glucoamylase complex. Both preparations contain a high-molecular weight polypeptide of approximately 250 kDa which in the case of sucrase-isomaltase could be identified by peptide mapping as a single-chain precursor not (yet) proteolytically processed to the final heterodimer. These first data on the mode of membrane anchoring of non-mammalian glycosidases indicate that they are synthesized, inserted into the membrane, and processed in ways similar to the mammalian enzymes. The fundamental unity between avian and mammalian sucrase-isomaltases suggests that the partial gene duplication of an ancestral isomaltase gene and the subsequent mutation of one of the active sites resulting in pro-sucrase-isomaltase has occurred prior to the separation of mammals from reptiles, i.e. more than 300 million years ago.     

Phylogenetic analysis reveals key residues in substrate hydrolysis in the isomaltase domain of sucrase-isomaltase and its role in starch digestion

BackgroundStarch constitutes one of the main sources of nutrition in the human diet and is broken down through a number of stages of digestion. Small intestinal breakdown of starch-derived substrates occurs through the mechanisms of small intestinal brush border enzymes, maltase-glucoamylase and sucrase-isomaltase. These enzymes each contain two functional enzymatic domains, and though they share sequence and structural similarities due to their evolutionary conservation, they demonstrate distinct substrate preferences and catalytic efficiency. The N-terminal isomaltase domain of sucrase-isomaltase has a unique ability to actively hydrolyze isomaltose substrates in contrast to the sucrase, maltase and glucoamylase enzymes.MethodsThrough phylogenetic analysis, structural comparisons and mutagenesis, we were able to identify specific residues that play a role in the distinct substrate preference. Mutational analysis and comparison with wild-type activity provide evidence that this role is mediated in part by affecting interactions between the sucrase and isomaltase domains in the intact molecule.ResultsThe sequence analysis revealed three residues proposed to play key roles in isomaltase specificity. Mutational analysis provided evidence that these residues in isomaltase can also affect activity in the partner sucrase domain, suggesting a close interaction between the domains.Major conclusionsThe sucrase and isomaltase domains are closely interacting in the mature protein. The activity of each is affected by the presence of the other.General Significance: There has been little experimental evidence previously of the effects on activity of interactions between the sucrase-isomaltase enzyme domains. By extension, similar interactions might be expected in the other intestinal α-glucosidase, maltase-glucoamylase.     

Biosynthetic processing of the oligosaccharide chains of cellular fibronectin

We have examined the maturation or processing of the oligosaccharides of cellular fibronectin in cultured chick embryo fibroblasts. Fibronectin was pulse-labeled with [2-³H]mannose or [³⁵S]methionine, and the turnover rates of carbohydrate and polypeptide portions of immunoprecipitated fibronectin were compared. The oligosaccharides on fibronectin were analyzed by gel electrophoresis for alterations in sensitivity to the enzyme endo-β-N-acetylglucosaminidase H, which specifically cleaves the ‘high-mannose’ class of asparagine-linked oligosaccharide. Incorporated mannose was removed only at early time points, suggesting that the structure of fibronectin oligosaccharides was altered due to processing.This possibility was confirmed by the analysis of glycopeptides generated by exhaustive pronase digestion. Two major glycopeptide structures were detected; their properties correspond to a ‘high-mannose’ oligosaccharide precursor and a ‘complex’ carbohydrate product. The precursor-product relationship of these two forms of oligosaccharide chains was demonstrated by pulse-chase labeling experiments. The precursor glycopeptide had an apparent size (M_r 2100) comparable to (Man)₉GlcNAc (M_r 2080), and was sensitive to endo-β-N-acetylglucosaminidase H; nearly all of the labeled mannose incorporated in a 10 min pulse was released from fibronectin glycopeptides by this enzyme. During a 90 min chase period, the glycopeptides became larger and increasingly resistent to endo-β-N-acetylglucosaminadase H cleavage. The final ‘complex’ or processed oligosaccharide structure contained approximately two-thirds less associated with the mature glycoprotein. They also indicate that the ‘complex’ structure is synthesized as a ‘high-mannose’ intermediate which is processed by the removal of mannose.     

Identification of a new gene in an operon for cellulose biosynthesis in Acetobacter xylinum

DNA sequencing of the region downstream of the cellulose synthase catalytic subunit gene of Acetobacter xylinum led to the identification of an open reading frame coding for a polypeptide of 86 kDa. The deduced amino acid sequence of this polypeptide matches from position 27 to 40 with the N-terminal amino acid sequence determined for a 93 kDa polypeptide that copurifies with the cellulose synthase catalytic subunit during purification of cellulose synthase. The cellulose synthase catalytic subunit gene and the gene encoding the 93 kDa polypeptide, along with other genes probably, are organized as an operon for cellulose biosynthesis in which the first gene is the catalytic subunit gene and the second gene codes for the 93 kDa polypeptide. The function of the 93 kDa polypeptide is not clear at present, however it appears to be tightly associated with the cellulose synthase catalytic subunit. Sequence analysis of the polypeptide shows that it is a membrane protein with a signal sequence at the N-terminal end and a transmembrane helix in the C-terminal region for anchoring it into the membrane.     

Structure-activity relationships of chlorinated benzenes as inducers of different forms of cytochrome P-450 in rat liver

Hexachlorobenzene (HCB) produced increases in ethoxyresorufin (ERR) O-deethylase, aryl hydrocarbon hydroxylase (AHH) and aminopyrine N-demethylase activities in rat liver microsomes which were intermediate between those produced by phenobarbital and 3,4-benzpyrene (BP). α-Naphthoflavone (ANF) selectively inhibited ERR activity in BP and HCB-induced microsomes (94% and 88%). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of liver microsomes indicated that HCB did not produce a detectable increase in a polypeptide with electrophoretic properties similar to those of purified cytochrome P-448 (M_r = 56 000). However, HCB did induce a polypeptide with M_r = 53 000 corresponding to one of two polypeptide bands induced by BP. This polypeptide may represent a second form of cytochrome P-448. Purification of HCB to remove possible dibenzo-p-dioxin impurities did not alter the ‘mixed-type’ induction produced by HCB. In contrast to HCB, all other chlorinated benzenes tested resembled phenobarbital as inducers.     

Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum

The gene for the catalytic subunit of cellulose synthase from Acetobacter xylinum has been cloned by using an oligonucleotide probe designed from the N-terminal amino acid sequence of the catalytic subunit (an 83 kDa polypeptide) of the cellulose synthase purified from trypsin-treated membranes of A. xylinum. The gene was located on a 9.5 kb HindIII fragment of A. xylinum DNA that was cloned in the plasmid pUC18. DNA sequencing of approximately 3 kb of the HindIII fragment led to the identification of an open reading frame of 2169 base pairs coding for a polypeptide of 80 kDa. Fifteen amino acids in the N-terminal region (positions 6 to 20) of the amino acid sequence, deduced from the DNA sequence, match with the N-terminal amino acid sequence obtained for the 83 kDa polypeptide, confirming that the DNA sequence cloned codes for the catalytic subunit of cellulose synthase which transfers glucose from UDP-glucose to the growing glucan chain. Trypsin treatment of membranes during purification of the 83 kDa polypeptide cleaved the first 5 amino acids at the N-terminal end of this polypeptide as observed from the deduced amino acid sequence, and also from sequencing of the 83 kDa polypeptide purified from membranes that were not treated with trypsin. Sequence analysis suggests that the cellulose synthase catalytic subunit is an integral membrane protein with 6 transmembrane segments. There is no signal sequence and it is postulated that the protein is anchored in the membrane at the N-terminal end by a single hydrophobic helix. Two potential N-glycosylation sites are predicted from the sequence analysis, and this is in agreement with the earlier observations that the 83 kDa polypeptide is a glycoprotein [13]. The cloned gene is conserved among a number of A. xylinum strains, as determined by Southern hybridization.     

Naturally occurring mutations in intestinal sucrase-isomaltase provide evidence for the existence of an intracellular sorting signal in the isomaltase subunit [published erratum appears in J Cell Biol 1991 Dec;115(5):following 1473]

Mutations in the sucrase-isomaltase gene can lead to the synthesis of transport-incompetent or functionally altered enzyme in congenital sucrase-isomaltase deficiency (CSID) (Naim, H. Y., J. Roth, E. Sterchi, M. Lentze, P. Milla, J. Schmitz, and H. P. Hauri. J. Clin. Invest. 82:667-679). In this paper we have characterized two novel mutant phenotypes of CSID at the subcellular and protein levels. The first phenotype revealed a sucrase-isomaltase protein that is synthesized as a single chain, mannose-rich polypeptide precursor (pro-SI) and is electrophoretically indistinguishable from pro-SI in normal controls. By contrast to normal controls, however, pro-SI does not undergo terminal glycosylation in the Golgi apparatus. Subcellular localization of pro-SI by immunoelectron microscopy revealed unusual labeling of the molecule in the basolateral membrane and no labeling in the brush border membrane thus indicating that pro-SI is missorted to the basolateral membrane. Mapping of biosynthetically labeled pro-SI with four epitope- and conformation-specific monoclonal antibodies suggested that conformational and/or structural alterations in the pro-SI protein have prevented posttranslational processing of the carbohydrate chains of the mannose-rich precursor and have lead to its missorting to the basolateral membrane. The second phenotype revealed two variants of pro-SI precursors that differ in their content of mannose-rich oligosaccharides. Conversion of these forms to a complex glycosylated polypeptide occurs at a slow rate and is incomplete. Unlike its counterpart in normal controls, pro-SI in this phenotype is intracellularly cleaved. This cleavage produces an isomaltase-like subunit that is transport competent and is correctly sorted to the brush border membrane since it could be localized in the brush border membrane by anti-isomaltase mAb. The sucrase subunit is not transported to the cell surface and is most likely degraded intracellularly. We conclude that structural features in the isomaltase region of pro-SI are required for transport and sorting of the sucrase-isomaltase complex.     

A component of the Xanthomonadaceae type IV secretion system combines a VirB7 motif with a N0 domain found in outer membrane transport proteins

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 Å X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7_XAC2622) and its interaction with VirB9. NMR solution studies show that residues 27–41 of the disordered flexible N-terminal region of VirB7_XAC2622 interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7_XAC2622 has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7_XAC2622 oligomerizes through interactions involving conserved residues in the N0 domain and residues 42–49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB7_XAC2622 oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring.     

Coagulation factor V exists uncomplexed in bovine plasma

Bovine coagulation factor V has been examined immunochemically to ascertain whether the coagulant polypeptide (h) with M_r = 290 000–330 000 is complexed in plasma with a second immunochemically distinct polypeptide (I₂) of M_r = 400 000. Antiserum containing antibodies to h and l₂ detects the l₂ polypeptide eluting earlier than the h chain on gel filtration of plasma with either added calcium or EDTA, consistent with the behavior of a higher molecular weight noninteracting species. An immobilized monospecific antibody to l₂ removes only the l₂ polypeptide from a purified factor V preparation containing both h and l₂. Moreover, while a monospecific antibody to the h chain was able to precipitate purified radioactively labelled h chain alone or mixed with plasma, the l₂ antibody was unable to precipitate radioactively labelled h chain even after attempted recombination of the h chain with l₂ present in plasma. These studies indicate that the l₂ polypeptide is not complexed to the h chain in a purified system or in plasma and reinforce the conclusion that factor V is a single polypeptide chain uncomplexed in plasma.     

Amino acid sequence of the small subunit of ribulose-1,5-bisphosphate carboxylase of Pisum sativum

The amino acid sequence of the small subunit of ribulose-1, 5-bisphosphate carboxylase from pea consists of a single polypeptide chain of 123 residues with a calculated MW of ca 14 480. The N-terminus was ‘ragged’ and both methionine and glutamine were determined in residue position 1. No heterogeneity was found even though two isofocussing variants were observed. The amino acid sequence confirms the nucleic acid sequence of cDNA of mRNA determined independently.     

Characterization of a 45-kDa polypeptide as the precursor of subunit 1 of cytochrome c oxidase in Neurospora crassa

In a previous paper (Van 't Sant, P., Mak, J.F.C. and Kroon, A.M. (1981) Eur. J. Biochem. 121, 21–26) we showed the existence of three elongated precursor proteins (45, 36 and 25 kDa) of mitochondrial translation products in Neurospora crassa. We presented some indications that the largest precursor could be related to subunit 1 of cytochrome c oxidase. Here we present conclusive evidence that the 45-kDa polypeptide is indeed this precursor by demonstrating that an immunodetectable 45-kDa polypeptide displays the same behaviour as the labeled 45-kDa precursor; both accumulate after long incubation with cycloheximide or by decreasing the temperature and both are not tightly membrane bound. Moreover the antibody against subunit 1 of cytochrome c oxidase also recognizes, in immunoadsorption experiments, besides subunit 1, the 45-kDa polypeptide accumulated by cycloheximide incubation. Furthermore, we developed a small scale purification of antibodies against subunit 1 of cytochrome c oxidase. By means of these purified antibodies it is demonstrated that the 45-kDa polypeptide and subunit 1 have corresponding antigenic determinants. Under the various conditions tested, all three precursors are less firmly membrane-bound than the mature subunits. Finally, it is observed that in short incubations in vivo, chloramphenicol inhibits the processing of the mitochondrially synthesized precursors, under conditions where mitochondrial translation is only partially inhibited.     

Crystal structure of the GABA(A)-receptor-associated protein, GABARAP

The GABA_A-receptor-associated protein (GABARAP) is a member of a growing family of intracellular membrane trafficking and/or fusion proteins and has been implicated in plasma membrane targeting and/or recycling of GABA_A receptors. GABARAP is localized on intracellular membranes such as the trans-Golgi network, binds to the γ 2 subunit of GABA_A receptors and interacts with microtubules and the N-ethylmaleimide-sensitive factor. We report the X-ray crystal structure of mammalian GABARAP at 2.0 Å resolution. GABARAP consists of an N-terminal basic helical region, which has been implicated in tubulin binding, and a core structure with a conserved ubiquitin-like fold. Consistent with the high extent of sequence conservation among GABARAP homologues from plants to mammals, one face of the core structure is absolutely conserved while the opposite face shows considerable divergence. These features are in agreement with the conserved surface mediating protein–protein interactions shared by all members of the family, whereas the non-conserved surface region may play specific roles, such as docking to particular membrane receptors.     

Biogenesis of mitochondria. Defective assembly of the proteolipid into the mitochondrial adenosine triphosphatase complex in an oli2 mit− mutant of Saccharomyces cerevisiae

A single mutation in the oli2 region of the mitochondrial DNA causes a charge alteration in a mitochondrially translated subunit of the mitochondrial ATPase (subunit 6; apparent M_r 20 000; apparent pI 6.9 and 7.1). This alteration leads to the defective assembly of the proteolipid subunit into the enzyme complex. The mutant, which is able to grow only very slowly by oxidative metabolism at 28°C offers new possibilities for studying the assembly of the membrane sector (F₀) into the mitochondrial ATPase complex and the role of subunit 6 in this process.     

Sucrase is an intramolecular chaperone located at the C-terminal end of the sucrase-isomaltase enzyme complex

The sucrase-isomaltase enzyme complex (pro-SI) is a type II integral membrane glycoprotein of the intestinal brush border membrane. Its synthesis commences with the isomaltase (IM) subunit and ends with sucrase (SUC). Both domains reveal striking structural similarities, suggesting a pseudo-dimeric assembly of a correctly folded and an enzymatically active pro-SI. The impact of each domain on the folding and function of pro-SI has been analyzed by individual expression and coexpression of the individual subunits. SUC acquires correct folding, enzymatic activity and transport competence and is secreted into the external milieu independent of the presence of IM. By contrast, IM persists as a mannose-rich polypeptide that interacts with the endoplasmic reticulum resident molecular chaperone calnexin. This interaction is disrupted when SUC is coexpressed with IM, indicating that SUC competes with calnexin for binding of IM. The interaction between SUC and the membrane-anchored IM leads to maturation of IM and blocks the secretion of SUC into the external milieu. We conclude that SUC plays a role as an intramolecular chaperone in the context of the pro-SI protein. To our knowledge all intramolecular chaperones so far identified are located at the N-terminal end. SUC is therefore the first C-terminally located intramolecular chaperone in mammalian cells.     

Limited proteol ysis of coupling factor-latent ATPase from Mycobacterium phlei. Effects of different enzymes and modifying agents

The activation of the coupling factor-latent ATPase enzyme by tryptic proteolysis may resemble the activation of many proenzymes by limited proteolysis. The beta (53 000 dalton) subunit of solubilized coupling factor-latent ATPase from Mycobacterium phlei was selectively lost in some trypsin-treated samples. Since a concomitant loss of ATPase activity was not observed, the beta subunit may not be essential for ATPase catalytic activity. Treatment of solublized coupling factor with chymotrypsin rapidly produced an A′-type (61 000 dalton) species from the native alpha (64 000 dalton) subunits with partial activation of the ATPase enzyme. Secondary chymotryptic cleavage yielded an A″-type (58 000 dalton) species and a less-active enzyme. Storage of fresh coupling factor samples at ?20°C in the presence of 4 mM MgCl₂ with several freeze-thaw cycles resulted in loss of ATPase activity without apparent change in alpha subunit structure. Storage at 4°C in the presence or absence of MgCl₂ both decreased ATPase activity and generated A′-type alpha subunit species. Since presence of phenylmethylsulfonyl fluoride prevented these changes, an unknown protease was suspected. The peptide bonds first cleaved by trypsin, chymotrypsin, and the unknown protease are all apparently located within the same small segment of alpha subunit polypeptide chain.