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PubMed1984

Substrate specificity of cholylglycine hydrolase for the hydrolysis of bile acid conjugates.

The substrate specificity of cholylglycine hydrolase has been investigated using bile acid conjugates with modifications in the steroid ring system, the side chain, or the amino acid moiety. Epimerization at C-3 and C-7 did not affect the activity of the enzyme while oxidation of the three nuclear hydroxyl groups reduced the affinity of the enzyme toward the substrate. Elongation of the side chain by one or three carbons inhibited enzyme activity. Conjugates prepared from C24 bile acids and analogs of taurine and glycine with one or two methylene groups were effectively hydrolyzed, whereas conjugates with a tertiary amide group completely resisted hydrolysis. Increasing the length of the bile acid side chain or using a bile acid conjugate with a tertiary amide group may produce compounds that will resist intestinal bacterial destruction.

Amidohydrolases↗
PubMed2002

Cloning, heterologous expression, and substrate specificities of protein farnesyltransferases from Trypanosoma cruzi and Leishmania major.

Chagas disease and leishmaniasis are tropical diseases caused by the protozoan parasites, Trypanosoma cruzi and Leishmania species, respectively. Protein farnesyltransferase (PFT) is being investigated as a target for anti-trypanosomatid agents because inhibitors of this enzyme are highly toxic to these parasites compared to mammalian cells. Here, we report the cloning of the alpha- and beta-subunit genes of PFT from T. cruzi and Leishmania major. The proteins encoded by these genes are considerably larger than those of mammalian PFTs due to the presence of a number of inserts of >25 amino acids that map to junctions between helical structural elements. These inserts are not part of the active site or the interface between the two subunits. Northern blots demonstrate expression of messenger RNA for the PFT subunits in both mammalian and insect life-cycle stages of these parasites. The T. cruzi, Trypanosoma brucei, and L. major PFTs were overexpressed in the Sf9 cell/baculovirus system as active enzyme forms. Kinetic studies with a panel of CALX-containing peptides with all 20 amino acids in the X-position show that trypanosomatid PFTs have similar substrate specificities and these are different from the mammalian PFT substrate specificity patterns.

Alkyl and Aryl Transferases↗
PubMed1990

Purification and characterization of an aminopeptidase from sperm of the sea urchin, Strongylocentrotus intermedius. Ca2(+)-dependent substrate specificity as a novel feature of the enzyme.

An aminopeptidase showing broad substrate specificity was purified to electrophoretic homogeneity from spermatozoa of the sea urchin, Strongylocentrotus intermedius. It is a single chain protein (Mr = 110,000) with an isoelectric point of 5.2 and shows the highest activity in a pH range between 7.0 and 7.5. Ni2+, Cu2+, Zn2+, and Hg2+, as well as 1,10-phenanthroline and p-chloromercuribenzoate, inhibit the enzyme irrespective of the substrates used, but Ca2+, Mn2+, Mg2+, and Co2+ modified the activity differently depending on the nature of the substrate. The effect of Ca2+ was most marked; it stimulated the activity toward some 4-methylcoumaryl-7-amide (MCA) substrates (for example leucine MCA), whereas it depressed the activity toward some other substrates such as arginine-MCA and lysine-MCA in a competitive manner. The rate of enzymatic hydrolysis determined for a mixture of leucine-MCA and arginine-MCA, in respect to the release of their common product (7-amino-4-methylcoumarin), was in good agreement with the value calculated on the assumption that these two substrates compete with each other for a single active site of the enzyme. Furthermore, the enzyme showed an identical Ki value for each of the competitive inhibitors examined, irrespective of the type of substrate. Ca2+ also influenced the activities toward various peptide substrates in a dual way similar to that observed on the MCA substrates. These results indicate that the sea urchin sperm aminopeptidase has an active site that alters its substrate preference depending on the Ca2+ concentration of the reaction medium.

Aminopeptidases↗
PubMed2006

Investigating the role of active site residues of Rhodotorula gracilis D-amino acid oxidase on its substrate specificity.

D-amino acid oxidase (DAAO) is a flavoprotein that catalyzes stereospecifically the oxidative deamination of D-amino acids. The wild-type DAAO is mainly active on neutral D-amino acids, while basic D-amino acids are poor substrates and the acidic ones are virtually not oxidized. To present a comprehensive picture of how the active site residues can modulate the substrate specificity a number of mutants at position M213, Y223, Y238, R285, S335, and Q339 were prepared in the enzyme from the yeast Rhodotorula gracilis. All DAAO mutants have spectral properties similar to those of the wild-type enzyme and are catalytically active, thus excluding an essential role in catalysis; a lower activity on neutral and basic amino acids was observed. Interestingly, an increase in activity and (k(cat)/K(m))(app) ratio on D-aspartate was observed for all the mutants containing an additional charged residue in the active site. The active site of yeast DAAO appears to be a highly evolved scaffold built up through evolution to optimize the oxidative deamination of neutral D-amino acids without limiting its substrate specificity. It is noteworthy, that introduction of a sole, additional, positively charged residue in the active site is sufficient to optimize the reactivity on acidic D-amino acids, giving rise to kinetic properties similar to those of D-aspartate oxidase.

Amino Acid Substitution↗
PubMed1988

Substrate specificity of ribosomal protein S6 kinase II from Xenopus eggs.

The substrate specificity of ribosomal protein S6 kinase II (S6 K II) from Xenopus eggs was evaluated using several protein substrates and a synthetic peptide corresponding to two phosphorylation sites in ribosomal protein S6. Previous studies had shown that S6 K II is unable to phosphorylate histones, casein, or phosvitin, proteins commonly used as substrates for protein kinases. In the present study S6 K II was found to phosphorylate with a significant stoichiometry rabbit skeletal muscle glycogen synthase, cardiac and skeletal muscle troponin I, and lamin C. In addition, the S6 peptide was phosphorylated by S6 K II to the same extent as observed with the catalytic subunit of cAMP-dependent protein kinase. Studies with oocytes undergoing progesterone-induced meiotic maturation and with activated or fertilized eggs revealed identical oscillations in both S6 and lamin C kinase activity. These results indicate that S6 K II does not have an absolute specificity for S6 in vitro. Therefore, since this enzyme is regulated during the cell cycle, it may phosphorylate several other proteins of interest during mitogenic stimulation.

Animals↗
PubMed1996

Homology modelling of rat kallikrein rK9, a member of the tissue kallikrein family: implications for substrate specificity and inhibitor binding.

The rat kallikrein rK9 is one of the six members of the rat tissue kallikrein family isolated to date. It is 84% identical to rK2 (tonin), and both proteinases are thought to have vasoconstrictive properties. Recently we have shown that rK9 and rK2 have distinct substrate specificities and sensitivities to inhibitors, despite their similar sequences. Unlike all other mammalian kallikrein-related proteinases, rK9 is resistant to inhibition by aprotinin. We have developed a 3-D model of rK9, based on the known X-ray structures of rK2, porcine kallikrein and bovine trypsin, to identify the structural features underlying this functional diversity. The final rK9 model is structurally similar to rK2, but variable regions surrounding the active site differ quite markedly from the reference proteins. The kallikrein loop, which differs from that in porcine kallikrein by a seven-residue insertion, has been generated de novo and subjected to simulated annealing to assess its influence on the restricted substrate specificity of these proteinases. The proposed conformation of the specificity pocket in rK9 differs from that of other serine proteinases, but it can still accommodate both aromatic and basic amino acid side chains at the substrate P1 position, thus explaining the dual chymotrypsin and trypsin-like activity of rK9. The electrostatic potentials of rK9 and aprotinin were calculated using the finite difference Poisson-Boltzmann method. They indicated a large positive region near the active site of rK9 not found in related proteinases because of positively charged residues at positions 61 and 65 in rK9. They generate a positive region, which overlaps a positive region in aprotinin, and may prevent aprotinin binding. A single mutation in aprotinin is suggested that might allow kallikrein rK9 inhibition by aprotinin. This model contributes significantly to our understanding of the structure-function relationships among proteinases of the tissue kallikrein family.

Amino Acid Sequence↗
PubMed2005

Profiling serine protease substrate specificity with solution phase fluorogenic peptide microarrays.

A novel microarray-based proteolytic profiling assay enabled the rapid determination of protease substrate specificities with minimal sample and enzyme usage. A 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized and microarrayed, along with fluorescent calibration standards, in glycerol nanodroplets on microscope slides. The arrays were then activated by deposition of an aerosolized enzyme solution, followed by incubation and fluorometric scanning. The specificities of human blood serine proteases (human thrombin, factor Xa, plasmin, and urokinase plasminogen activator) were examined. The arrays provided complete maps of protease specificity for all of the substrates tested and allowed for detection of cooperative interactions between substrate subsites. The arrays were further utilized to explore the conservation of thrombin specificity across species by comparing the proteolytic fingerprints of human, bovine, and salmon thrombin. These enzymes share nearly identical specificity profiles despite approximately 390 million years of divergent evolution. Fluorogenic substrate microarrays provide a rapid way to determine protease substrate specificity information that can be used for the design of selective inhibitors and substrates, the study of evolutionary divergence, and potentially, for diagnostic applications.

Amino Acid Motifs↗
PubMed1998

Mutual conversion of fatty-acid substrate specificity by a single amino-acid exchange at position 527 in P-450Cm2 and P-450Alk3A.

The two eukaryotic fatty-acid hydroxylases P-450Cm2 and P-450Alk3A, which represent CYP52A4 variants naturally occurring in the yeast Candida maltosa, were characterized with respect to their substrate specificity. Whereas P-450Cm2 was found to catalyse lauric acid omega-hydroxylation with greater efficiency, P-450Alk3A had higher palmitic acid turnover numbers compared to P-450Cm2, resulting in ratios of lauric acid to palmitic acid turnover rates of nearly 11 and 3 for P-450Cm2 and P-450Alk3A, respectively. As shown by means of chimeric enzymes and site-directed mutagenesis, the key residue determining these differences in substrate specificity was found to be a single amino acid at position 527. Interestingly, the mutual exchange of valine (P-450Cm2) and leucine (P-450Alk3A) led to a direct transposition of specificity, suggesting that amino acids at this site may determine the efficiency of fatty-acid hydroxylation relatively independently of other active-site residues. This was further supported by the finding that P-450Cm2 and P-450Alk3A with methionine at position 527 displayed almost identical hydroxylation activities. Moreover, methionine to leucine substitutions at the corresponding alignment position in P-450Cm1 (CYP52A3), P-450Alk2A (CYP52A5) and P-450Alk5A (CYP52A9) altered the fatty-acid specificity of these enzymes. In comparison to the structure of the bacterial P-450BM3 (CYP102), we propose that the amino acid at position 527 may serve to close the substrate-binding pocket near to the haem in the fatty-acid-omega-hydroxylating P-450 of the CYP52 family.

Amino Acid Sequence↗
PubMedDate not supplied

Glucocorticoids upregulate intestinal nutrient transport in a time-dependent and substrate-specific fashion.

Glucocorticoids mediate skeletal muscle proteolysis during critical illness to provide substrates for hepatic acute-phase protein synthesis and gluconeogenesis. The effects of hypercortisolemia on splanchnic substrate uptake are not well defined. This study characterizes intestinal nutrient transport in response to acute elevations of plasma glucocorticoid levels. New Zealand White rabbits were randomized to receive either dexamethasone (2 mg/kg intramuscularly) or vehicle and were killed 8, 16, or 24 hours after steroid treatment. Brush-border membrane vesicles were prepared from pooled small intestinal mucosa and the uptake of tritiated substrates was quantified. Serum insulin-like growth factor 1 (IGF-1) levels, mucosal DNA content, and mucosal morphology were determined. Glucocorticoids increased glucose and leucine uptake at 8 hours (80% and 24%, respectively) and 24 hours (147% and 50%, respectively). Glutanmine, alanine, and arginine transport increased by 42%, 96%, and 236%, respectively, at 24 hours. Sodium-independent transport (diffusion) of all substrates was increased by 240% by dexamethasone treatment at 24 hours. Mucosal DNA content increased by 32%, whereas microvillus heights decreased by 27% at 24 hours. No effects were noted on IGF-1 levels or gross villus heights. Glucocorticoids acutely accelerate intestinal nutrient transport in a time-related and substrate-specific fashion. Although the mechanism of glucocorticoid action remains unclear, both genomic and plasma membrane effects are implicated.

Alanine↗
PubMed2005

Alteration of substrate specificity of aspartase by directed evolution.

Aspartase (l-aspartate ammonia-lyase, EC 4.3.1.1), which catalyzes the reversible deamination of l-aspartic acid to yield fumaric acid and ammonia, is highly selective towards l-aspartic acid. We screened for enzyme variants with altered substrate specificity by a directed evolution method. Random mutagenesis was performed on an Escherichia coli aspartase gene (aspA) by error-prone PCR to construct a mutant library. The mutant library was introduced to E. coli and the transformants were screened for production of fumaric acid-mono amide from l-aspartic acid-alpha-amide. Through the screening, one mutant, MA2100, catalyzing deamination of l-aspartic acid-alpha-amide was achieved. Gene analysis of the MA2100 mutant indicated that the mutated enzyme had a K327N mutation. The characteristics of the mutated enzyme were examined. The optimum pH values for the l-aspartic acid and l-aspartic acid-alpha-amide of the mutated enzyme were pH 8.5 and 6.0, respectively. The K(m) value and V(max) value for the l-aspartic acid of the mutated enzyme were 28.3 mM and 0.26 U/mg, respectively. The K(m) value and V(max) value for the l-aspartic acid-alpha-amide of the mutated enzyme were 1450 mM and 0.47 U/mg, respectively. This is the first report describing the alteration of the substrate specificity of aspartase, an industrially important enzyme.

Aspartate Ammonia-Lyase↗
PubMed1999

Multisite-specific tRNA:m5C-methyltransferase (Trm4) in yeast Saccharomyces cerevisiae: identification of the gene and substrate specificity of the enzyme.

Several genes encoding putative RNA:5-methylcytidine-transferases (m5C-transferases) from different organisms, including yeast, have been identified by sequence homology with the recently identified 16S rRNA:m5C967-methyltransferase (gene SUN) from Escherichia coli. One of the yeast ORFs (YBL024w) was amplified by PCR, inserted in the expression vector pET28b, and the corresponding protein was hyperexpressed in E. coli BL21 (DE3). The resulting N-terminally His6-tagged recombinant Ybl024p was purified to apparent homogeneity by one-step affinity chromatography on Ni2+-NTA-agarose column. The activity and substrate specificity of the purified Ybl024p were tested in vitro using T7 transcripts of different yeast tRNAs as substrates and S-adenosyl-L-methionine as a donor of the methyl groups. The results indicate that yeast ORF YBL024w encodes S-adenosyl-L-methionine-dependent tRNA: m5C-methyltransferase that is capable of methylating cytosine to m5C at several positions in different yeast tRNAs and pre-tRNAs containing intron. Modification of tRNA occurs at all four positions (34, 40, 48, and 49) at which m5C has been found in yeast tRNAs sequenced so far. Disruption of the ORF YBL024w leads to the complete absence of m5C in total yeast tRNA. Moreover no tRNA:m5C-methyltransferase activity towards all potential m5C methylation sites was detected in the extract of the disrupted yeast strain. These results demonstrate that the protein product of a single gene is responsible for complete m5C methylation of yeast tRNA. Because this newly characterized multisite-specific modification enzyme Ybl024p is the fourth tRNA-specific methyltransferase identified in yeast, we suggest designating it as TRM4, the gene corresponding to ORF YBL024w.

Amino Acid Sequence↗
PubMed2000

Display of active subtilisin 309 on phage: analysis of parameters influencing the selection of subtilisin variants with changed substrate specificity from libraries using phosphonylating inhibitors.

Many attempts have been made to endow enzymes with new catalytic activities. One general strategy involves the creation of random combinatorial libraries of mutants associated with an efficient screening or selection scheme. Phage display has been shown to greatly facilitate the selection of polypeptides with desired properties by establishing a close link between the polypeptide and the gene that encodes it. Selection of phage displayed enzymes for new catalytic activities remains a challenge. The aim of this study was to display the serine protease subtilisin 309 (savinase) from Bacillus lentus on the surface of filamentous fd phage and to develop selection schemes that allow the extraction of subtilisin variants with a changed substrate specificity from libraries. Subtilisins are produced as secreted preproenzyme that mature in active enzyme autocatalytically. They have a broad substrate specificity but exhibit a significant preference for hydrophobic residues and very limited reactivity toward charged residues at the P4 site in the substrate. Here, we show that savinase can be functionally displayed on phage in the presence of the proteic inhibitor CI2. The free enzyme is released from its complex with CI2 upon addition of the anionic detergent LAS. The phage-enzyme can be panned on streptavidin beads after labelling by reaction with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-l-Ala-l-Ala-l-P ro-Phe(P)-diphenyl ester. Reactions of libraries, in which residues 104 and 107 forming part of the S4 pocket have been randomised, with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-alpha-l-Lys-l-A la-l-Pro-Phe(P)-diphenylester allowed us to select enzymes with increased specific activity for a substrate containing a lysine in P4. Parameters influencing the selection as for instance the efficiency of maturation of mutant enzymes in libraries have been investigated.

Alkanesulfonic Acids↗
PubMed2004

Substrate specificity of the Leishmania donovani myo-inositol transporter: critical role of inositol C-2, C-3 and C-5 hydroxyl groups.

Inositol is an essential precursor for the formation of glycosyl-phosphatidylinositol (GPI)-anchors found in the majority of surface molecules in trypanosomatids, in addition to its requirement for phoshatidylinositol signal transduction pathways. In Leishmania donovani, high-affinity inositol transport is catalyzed by the active myo-inositol/H+ transporter MIT, which is driven by a proton gradient across the parasite membrane. We have characterized the substrate specificity and pharmacology of L. donovani MIT in vitro and in promastigote cultures. High substrate specificity of myo-inositol transport was shown in competition studies with 14 different monosaccharides and MIT function was unaffected by the structurally similar pentose sugars or hexoses. L-Fucose and D-xylose, both inhibitors of the Na+-dependent inositol transport system in the human host, did not affect MIT transport function in the parasite. Competition studies with eight different inositol isomers revealed that proton bonds between the C-2, C-3 and C-5 hydroxyl groups of myo-inositol and the transporter protein played a critical role for substrate recognition, and the C-3 hydroxyl oxygen appears to act as an electron donor to form an H-bond with a positive charge of the MIT permease. The cytotoxic inositol analogue 3-fluoro-myo-inositol was recognized by MIT with similar affinity as myo-inositol and showed an IC50 value of 42 +/- 8 microM in L. donovani cultures. Finally, substrate affinities of MIT revealed apparent Km values of 84 +/- 8 microM for myo-inositol and 5.4 +/- 0.9 nM for H+, equal pH 8.27 + 0.08, suggesting that the L. donovani myo-inositol/H+ symporter is fully activated at physiological pH in the sandfly midgut or macrophage phagolysosome. We conclude that Leishmania MIT constitutes an attractive target for delivery of cytotoxic inositol analogues and differs significantly from the sodium-coupled myo-inositol transport system of the human host.

Animals↗
PubMed1993

Substrate specificity of the gastrin-amidating enzyme.

As is the case with many other peptide hormones of the brain and gut, gastrin requires a carboxyl-terminal amide moiety for optimal biological activity. In the structure of progastrin, the carboxyl-terminal Phe of gastrin is followed by the sequence Gly93-Arg94-Arg95, which must be processed sequentially by an endoprotease, a carboxypeptidase, and an amidating enzyme to produce amidated bioactive gastrin. To examine the molecular determinants of peptide amidation in vivo, we mutated the wild-type Gly93 residue of progastrin to Ala93 and Ser93 and expressed the three progastrin DNAs in GH3 and MTC 6-23 endocrine cell lines. Although substantial quantities of amidated gastrin were seen in cells expressing wild-type progastrin, replacement of Gly93 with Ala93 completely abolished production of amidated gastrin when the cells were incubated in standard medium containing only L-alanine. In a similar fashion, cells expressing [Ser93]progastrin also demonstrated no production of amidated gastrin. When cells expressing [Ala93]- or [Ser93]progastrin were incubated in the presence of 1 mg/ml D-alanine or D-serine, respectively, a small but consistent amount of amidated gastrin production was detected (< 1% of wild type). These data lead us to conclude that the amidating enzyme has a rigid substrate specificity for a glycine-extended precursor. Furthermore, this in vivo substrate specificity confirms the importance of the pro-S-alpha-hydrogen of the carboxyl-terminal glycine for enzyme-substrate recognition.

Amides↗
PubMed2000

MALDI-TOF mass spectrometry analysis of substrate specificity of lebetase, a direct-acting fibrinolytic metalloproteinase from Vipera lebetina snake venom.

Lebetase is a direct-acting fibrinolytic zinc metalloendopeptidase related in amino acid sequence to reprolysins which include both hemorrhagic and non-hemorrhagic proteinases. Despite apparent structural similarities, fibrinolytic and hemorrhagic proteinases differ significantly in substrate specificity. In this study, we have examined the activity of lebetase I against biologically active peptides (bradykinin, kallidin, substance P) and 6-10 amino acid residues containing peptides synthesized according to cleavage regions of alpha(2)-macroglobulin, pregnancy zone protein (PZP) and fibrinogen. Lebetase was found to have no activity against studied hexapeptides. Surprisingly, the best substrates for lebetase were substance P, and peptide fragment of PZP, both were cleaved at position Pro-Gln. Identification of the hydrolysis products of 15 peptides by MALDI-TOF mass spectrometry analysis indicates that lebetase possesses broad substrate specificity. The MALDI-TOF MS technique was proven to be highly efficient for the recovery and identification of the peptides released by lebetase hydrolysis.

Animals↗
PubMed2003

Substrate specificity of mouse aldo-keto reductase AKR7A5.

We have determined the substrate specificity of a mouse aldo-keto reductase (AKR) AKR7A5, an enzyme that is similar to rat aflatoxin aldehyde reductase (AKR7A1) and to human brain succinic semialdehyde reductase (AKR7A2). Previously, we have shown that the mouse enzyme is present in a range of tissues including liver, kidney, testis and brain, and is able to reduce several carbonyl compounds, including succinic semialdehyde, 2-carboxybenzaldehyde, 4-nitrobenzaldehyde and 9,10-phenanthrenequinone [FEBS Lett. 523 (2002) 213]. It has been suggested that it may represent the mouse equivalent of human succinic semialdehyde reductase which is responsible for the biosynthesis of gamma-hydroxybutyrate. In this study, we show that the enzyme is also able to reduce other aromatic aldehydes such as 4-chloro-3-nitrobenzaldehyde, and 3-nitrobenzaldehyde, and has particular high specific activity towards dicarbonyls such as acenapthenequinone, 2,3-bornanedione (camphorquinone), and phenylglyoxal. It has low specific activity towards ketones, and alpha,beta-unsaturated carbonyls such as acrolein and 4-hydroxynonal. The enzyme is inhibited by several compounds including quercitin, ethacrynic acid, indomethacin and sodium valproate. Developing selective inhibitors may lead to a means of modifying the activity of the enzyme in vivo.

Alcohol Oxidoreductases↗
PubMed2005

Two rhizobial strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, encode haloalkane dehalogenases with novel structures and substrate specificities.

Haloalkane dehalogenases are key enzymes for the degradation of halogenated aliphatic pollutants. Two rhizobial strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, have open reading frames (ORFs), mlr5434 and blr1087, respectively, that encode putative haloalkane dehalogenase homologues. The crude extracts of Escherichia coli strains expressing mlr5434 and blr1087 showed the ability to dehalogenate 18 halogenated compounds, indicating that these ORFs indeed encode haloalkane dehalogenases. Therefore, these ORFs were referred to as dmlA (dehalogenase from Mesorhizobium loti) and dbjA (dehalogenase from Bradyrhizobium japonicum), respectively. The principal component analysis of the substrate specificities of various haloalkane dehalogenases clearly showed that DbjA and DmlA constitute a novel substrate specificity class with extraordinarily high activity towards beta-methylated compounds. Comparison of the circular dichroism spectra of DbjA and other dehalogenases strongly suggested that DbjA contains more alpha-helices than the other dehalogenases. The dehalogenase activity of resting cells and Northern blot analyses both revealed that the dmlA and dbjA genes were expressed under normal culture conditions in MAFF303099 and USDA110 strain cells, respectively.

Alphaproteobacteria↗
PubMed2005

Molecular cloning and expression of human ST6GalNAc III: restricted tissue distribution and substrate specificity.

We isolated human ST6GalNAc III cDNA clones. The typical cDNA clones predicted a type II membrane protein of 305 amino acids with a short cytoplasmic transmembrane domain of sixteen amino acids and a catalytic domain of 280 amino acids. A short form clone predicted a protein of 240 amino acids lacking 65 amino acids including the transmembrane portion. The alternative usage of the second exon seemed to generate these two transcripts. Both had two common regions found among sialyltransferases cloned so far, i.e. sialyl motif L and sialyl motif S. Alignments of human, mouse and rat orthologs indicated that high homologies, i.e. 85-95% identity among these species at amino acid levels. We analyzed the expression pattern and substrate specificity of the product, demonstrating a very restricted expression pattern and a high substrate specificity. Northern blotting revealed that hST6GalNAc III is expressed in kidney and brain as a single band at 3.2 kb. In enzyme assay of the long form, the transfer of sialic acid onto alpha2,3-sialylated acceptor substrates, i.e. GM1b and sialyl lactotetraosylceramide, was observed. hST6GalNAc III also showed sialyltransferase activity toward O-glycans (but not N-glycans) in fetuin.

Alternative Splicing↗