Cloning and characterization of a third type of human alpha-amylase gene, AMY2B

We have previously reported concerning the existence of a third type of human alpha-amylase gene, AMY3 [Emi et al., Gene 62 (1988) 229-235; Tomita et al., Gene 76 (1989) 11-18], which is expressed in a lung carcinoid tissue, and differs in nucleotide sequence from the two previously characterized human alpha-amylase genes coding for salivary and pancreatic isozymes, termed AMY1 and AMY2, respectively. Here, we rename this gene AMY2B to coincide with the designation by Gumucio et al. [Mol. Cell Biol. 8 (1988) 1197-1205] and describe its genetic properties as revealed by sequencing studies. It consists of ten major exons whose sequences are highly homologous to those of AMY1 and AMY2. Not only the exons, but also most of the introns seem to be highly conserved, as judged from physical mapping data. The AMY2B gene identified from mRNA in a lung carcinoid tissue has at least two additional untranslated exons in its 5' region; hence the promoter lies far upstream relative to the other two AMY genes.     

SNP analysis of AMY2 and CTSL genes in Litopenaeus vannamei and Penaeus monodon shrimp

Genetic studies in shrimp have focused on disease, with production traits such as growth left unexamined. Two shrimp species, Litopenaeus vannamei and Penaeus monodon, which represent the majority of US shrimp imports, were selected for single nucleotide polymorphism (SNP) discovery in alpha-amylase (AMY2) and cathepsin-l (CTSL), both candidate genes for growth. In L. vannamei, four SNPs were found in AMY2 and one SNP was found in CTSL. In P. monodon, one SNP was identified in CTSL. The CTSL gene was mapped to linkage group 28 of P. monodon using the female map developed with the Australian P. monodon mapping population. Association analyses for the AMY2 and CTSL genes with body weight (BW) were performed in two L. vannamei populations. While neither gene was found to be significantly associated with BW in these populations, there was a trend in one population towards higher BW for allele G of CTSL SNP C681G.     

Salivary protein polymorphism in Kenya: evidence for a new AMY1 allele

Salivary protein polymorphism was studied in 200 schoolboys, mainly Kisii and Luo from Kenya, East Africa. The frequencies of PR, PA, DB, PB and AMY1 genes were as follows: PR*1: 0.66, PA*(+): 0.18, DB*(+): 0.55, PB*2: 0.12, AMY1*A2: 0.008, AMY1*E: 0.03. These frequencies were compared with other population data, in particular from West African and US Negroes. The most interesting finding with respect to the gene frequencies is the low PB*2 frequency and the absence of AMY1*3 in Kenya. Furthermore, a new phenotype in the AMY1 system was described which suggests the presence of an allele with an estimated frequency of 0.02.     

Effects of calcium ion concentration on starch hydrolysis of barley alpha-amylase isozymes

Barley alpha-amylase genes, amy1 and amy2, were separately cloned into the expression vector of pPICZalphaA and recombinant Pichia strains were established by homologous recombination. Both AMYs from Pichia shared almost identical hydrolysis patterns on short maltooligosaccharides to result in glucose, maltose, or maltotriose. Against insoluble blue starch, AMY1 showed the highest activity at 0.1-5 mM calcium concentration, whereas 15-20 mM was optimal for AMY2. On the hydrolysis of soluble starch, unexpectedly, there was no significant difference between AMYs with increase of calcium. However, the relative activity on various starch substrates was significantly different between AMYs, which supports that the isozymes are clearly distinguished from each other on the basis of their unique preferences for substrates.     

Distribution of AMY2 polymorphism in S. Tomé and Príncipe (West Africa)

The genetic polymorphism of AMY2 was studied in the population of S. Tomé and Príncipe (West Africa) using agarose gel electrophoresis. AMY2 frequencies are reported for the first time in a subSaharian population. The gene frequencies found were: AMY2*1=0.948, AMY2*3=0.052 (N=173).     

Amylase activity is associated with AMY2B copy numbers in dog: implications for dog domestication,diet and diabetes

High amylase activity in dogs is associated with a drastic increase in copy numbers of the gene coding for pancreatic amylase, AMY2B, that likely allowed dogs to thrive on a relatively starch‐rich diet during early dog domestication. Although most dogs thus probably digest starch more efficiently than do wolves, AMY2B copy numbers vary widely within the dog population, and it is not clear how this variation affects the individual ability to handle starch nor how it affects dog health. In humans, copy numbers of the gene coding for salivary amylase, AMY1, correlate with both salivary amylase levels and enzyme activity, and high amylase activity is related to improved glycemic homeostasis and lower frequencies of metabolic syndrome. Here, we investigate the relationship between AMY2B copy numbers and serum amylase activity in dogs and show that amylase activity correlates with AMY2B copy numbers. We then describe how AMY2B copy numbers vary in individuals from 20 dog breeds and find strong breed‐dependent patterns, indicating that the ability to digest starch varies both at the breed and individual level. Finally, to test whether AMY2B copy number is strongly associated with the risk of developing diabetes mellitus, we compare copy numbers in cases and controls as well as in breeds with varying diabetes susceptibility. Although we see no such association here, future studies using larger cohorts are needed before excluding a possible link between AMY2B and diabetes mellitus.     

Population genetic studies of the pancreatic amylase (AMY2, E.C. 3.2.1.1) in Bulgaria

The pancreatic amylase (AMY2, E.C. 3.2.1.1) polymorphism has been studied in 2346 individuals from south-central and south-eastern Bulgaria. The allele frequencies have been determined as AMY2*1 = 0.9520 and AMY2*2 = 0.0480. The neighbor joining tree of seven subpopulations revealed only small genetic distances. Compared with other populations, the Bulgarian sample clustered with samples from Romania, Hungary, Germany and Switzerland, with larger distances to Albania, Greece and Macedonia.     

Salivary enzyme polymorphisms (Set, Sgd and AMY1) in the Galician population

The genetic polymorphism of three salivary enzymes (esterase, glucose-6-phosphate dehydrogenase and amylase) was studied in 580 autochthonous individuals from the Galician population (North-West Spain). The gene frequencies obtained were: SetF = 0.4036, SetS = 0.5964; Sgd1 = 0.7828, Sgd2 = 0.2172; AMY11 = 0.9319, AMY21 = 0.0495, AMY31 = 0.0186. Evidence of genetic intrapopulational heterogeneity was found for Set and Sgd loci. An alternative method for AMY1 typing by means of isoelectric focusing is proposed which allows the use of long-term stored saliva samples.     

Copy number variations in the amylase gene (AMY2B) in Japanese native dog breeds

A recent study suggested that increased copy numbers of the AMY2B gene might be a crucial genetic change that occurred during the domestication of dogs. To investigate AMY2B expansion in ancient breeds, which are highly divergent from modern breeds of presumed European origins, we analysed copy numbers in native Japanese dog breeds. Copy numbers in the Akita and Shiba, two ancient breeds in Japan, were higher than those in wolves. However, compared to a group of various modern breeds, Akitas had fewer copy numbers, whereas Shibas exhibited the same level of expansion as modern breeds. Interestingly, average AMY2B copy numbers in the Jomon‐Shiba, a unique line of the Shiba that has been bred to maintain their appearance resembling ancestors of native Japanese dogs and that originated in the same region as the Akita, were lower than those in the Shiba. These differences may have arisen from the earlier introduction of rice farming to the region in which the Shiba originated compared to the region in which the Akita and the Jomon‐Shiba originated. Thus, our data provide insights into the relationship between the introduction of agriculture and AMY2B expansion in dogs.     

Identification of a novel alpha-amylase by expression of a newly cloned human amy3 cDNA in yeast

A novel amylase gene (amy3) that differs in nucleotide sequence from salivary amylase gene (amy1) and pancreatic amylase gene (amy2) has been described [Tomita et al., Gene 76 (1989) 11-18], but whether this gene can ever code for an active enzyme has not been shown. We prepared cDNA of this gene from an mRNA obtained from lung carcinoid tissue, and expressed it in Saccharomyces cerevisiae under the control of an acid phosphatase promoter. The product was secreted into culture media, and showed enzymatic activity, demonstrating that this novel alpha-amylase gene (amy3) can code for a functional isozyme. We purified this enzyme, and compared its biological properties with those of salivary and pancreatic human amylases similarly expressed in yeast. We observed that the novel amylase isozyme is more heat-sensitive than others, and that its substrate specificity is different from the other two isozymes.     

Genetic coadaptation of the amylase gene system in Drosophila melanogaster: evidence for the selective advantage of the lowest AMY activity and of its epistatic genetic background

In natural populations of Drosophila melanogaster, an amylase isozyme with the lowest alpha-amylase activity (AMY(1,1)) is predominant. To evaluate the selective significance of AMY(1,1) and its regulatory factor(s), we examined selection experiments in laboratory populations on two distinct food environments. After 300 generations, AMY(1,1) became predominant (89%) in a glucose (a product of AMY)-rich environment, while an isozyme with higher alpha-amylase activity, AMY(1,6), became predominant (83%) in a starch (substrate)-rich environment. We found that the identical alleles of the amylase (Amy) gene, which encodes each of AMY(1,1) and AMY(1,6), were shared between the two populations in the different food environments, employing the nucleotide sequencing of the duplicated Amy genes. Nevertheless, AMY(1,6) homozygotes selected in the starch-rich environment had a twofold higher AMY enzyme activity than those selected in the glucose-rich environment, suggesting a coadaptation of the coding region and its regulatory factor(s) on the genetic background. Such a difference in AMY enzyme activity was not detected between AMY(1,1) homozygotes, suggesting that the effect of the genetic background is epistatic. Our results indicate that natural selection is working on the Amy gene system as a whole for flies to adapt to the various food environments of local populations.     

Regulation of Leaf Starch Degradation by Abscisic Acid Is Important for Osmotic Stress Tolerance in Plants

Starch serves functions that range over a timescale of minutes to years, according to the cell type from which it is derived. In guard cells, starch is rapidly mobilized by the synergistic action of β-AMYLASE1 (BAM1) and α-AMYLASE3 (AMY3) to promote stomatal opening. In the leaves, starch typically accumulates gradually during the day and is degraded at night by BAM3 to support heterotrophic metabolism. During osmotic stress, starch is degraded in the light by stress-activated BAM1 to release sugar and sugar-derived osmolytes. Here, we report that AMY3 is also involved in stress-induced starch degradation. Recently isolated Arabidopsis thaliana amy3 bam1 double mutants are hypersensitive to osmotic stress, showing impaired root growth. amy3 bam1 plants close their stomata under osmotic stress at similar rates as the wild type but fail to mobilize starch in the leaves. ¹⁴C labeling showed that amy3 bam1 plants have reduced carbon export to the root, affecting osmolyte accumulation and root growth during stress. Using genetic approaches, we further demonstrate that abscisic acid controls the activity of BAM1 and AMY3 in leaves under osmotic stress through the AREB/ABF-SnRK2 kinase-signaling pathway. We propose that differential regulation and isoform subfunctionalization define starch-adaptive plasticity, ensuring an optimal carbon supply for continued growth under an ever-changing environment.     

Rethinking the starch digestion hypothesis for AMY1 copy number variation in humans

Alpha‐amylase exists across taxonomic kingdoms with a deep evolutionary history of gene duplications that resulted in several α‐amylase paralogs. Copy number variation (CNV) in the salivary α‐amylase gene (AMY1) exists in many taxa, but among primates, humans appear to have higher average AMY1 copies than nonhuman primates. Additionally, AMY1 CNV in humans has been associated with starch content of diets, and one known function of α‐amylase is its involvement in starch digestion. Thus high AMY1 CNV is considered to result from selection favoring more efficient starch digestion in the Homo lineage. Here, we present several lines of evidence that challenge the hypothesis that increased AMY1 CNV is an adaptation to starch consumption. We observe that α‐ amylase plays a very limited role in starch digestion, with additional steps required for starch digestion and glucose metabolism. Specifically, we note that α‐amylase hydrolysis only produces a minute amount of free glucose with further enzymatic digestion and glucose absorption being rate‐limiting steps for glucose availability. Indeed α‐amylase is nonessential for starch digestion since sucrase‐isomaltase and maltase‐glucoamylase can hydrolyze whole starch granules while releasing glucose. While higher AMY1 CN and CNV among human populations may result from natural selection, existing evidence does not support starch digestion as the major selective force. We report that in humans α‐amylase is expressed in several other tissues where it may have potential roles of evolutionary significance.     

Degradation of Glucan Primers in the Absence of Starch Synthase 4 Disrupts Starch Granule Initiation in Arabidopsis

Arabidopsis leaf chloroplasts typically contain five to seven semicrystalline starch granules. It is not understood how the synthesis of each granule is initiated or how starch granule number is determined within each chloroplast. An Arabidopsis mutant lacking the glucosyl-transferase, STARCH SYNTHASE 4 (SS4) is impaired in its ability to initiate starch granules; its chloroplasts rarely contain more than one large granule, and the plants have a pale appearance and reduced growth. Here we report that the chloroplastic α-amylase AMY3, a starch-degrading enzyme, interferes with granule initiation in the ss4 mutant background. The amy3 single mutant is similar in phenotype to the wild type under normal growth conditions, with comparable numbers of starch granules per chloroplast. Interestingly, the ss4 mutant displays a pleiotropic reduction in the activity of AMY3. Remarkably, complete abolition of AMY3 (in the amy3 ss4 double mutant) increases the number of starch granules produced in each chloroplast, suppresses the pale phenotype of ss4, and nearly restores normal growth. The amy3 mutation also restores starch synthesis in the ss3 ss4 double mutant, which lacks STARCH SYNTHASE 3 (SS3) in addition to SS4. The ss3 ss4 line is unable to initiate any starch granules and is thus starchless. We suggest that SS4 plays a key role in granule initiation, allowing it to proceed in a way that avoids premature degradation of primers by starch hydrolases, such as AMY3.     

Expression of cDNAs encoding barley alpha-amylase 1 and 2 in yeast and characterization of the secreted proteins

Amylolytic strains of the yeast, Saccharomyces cerevisiae, were constructed by transformation with expression plasmids containing cDNAs encoding either AMY1 (clone E) or AMY2 (clone pM/C). The alpha-amylases were efficiently secreted into the culture medium directed by their own signal peptides. When clone E without its 5'-noncoding region was expressed from the yeast PGK promoter, AMY1 was produced as 1% of total cell protein and was thus the major protein secreted, whereas a similar construct derived from pM/C produced much less AMY2. This level is the highest reported for a plant protein secreted by yeast as mediated by the endogenous signal peptide. Production of AMY1 increased 25-fold when the 5'-noncoding part of clone E which contains a 12-bp dG.dC homopolymer tail had been removed. Moreover, expression was one to two orders of magnitude higher when genes encoding AMY1 or AMY2 were inserted between promoter and terminator of the yeast PGK gene in comparison to expression directed from the ADC1 or GAL1 promoters. Recombinant AMY1 and AMY2 had the same Mr and N-terminal sequence as the corresponding barley malt enzymes. Furthermore, none of the enzymes were found to be N-glycosylated. Isoelectric focusing indicated that transformed yeast cells secreted one major form of AMY2 and four dominant forms of AMY1. One AMY1 form corresponded to one of the major forms found in malt while the others, having either low activity or unusually high pI, probably reflect inefficient/incorrect processing. Enzyme kinetic properties and pH activity-dependence of recombinant AMY2 were essentially identical to those of malt AMY2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Probable genetic linkage between autosomal dominant retinitis pigmentosa (RP) and amylase (AMY2): evidence of an RP locus on chromosome 1.

A linkage analysis is reported for three branches of a single family segregating for autosomal dominant retinitis pigmentosa. A statistically significant lod score of 3.9 is obtained for the RP locus and AMY2 at a recombination frequency of 1%. This linkage indicates that the RP locus is on the no. 1 chromosome since the AMY2 locus has been placed on the short arm of 1. Lod scores are reported for four other loci on chromosome 1; none of these achieve statistical significance. Analyses are reported for 23 additional autosomal markers and close linkage with RP can be excluded for a number of these.     

Detection of human urinary alpha-amylase encoded by the AMY2B gene using a fluorogenic substrate, FG5P.

The existence of alpha-amylase (HXA) encoded by alpha-amylase gene AMY2B in healthy humans was examined using a fluorogenic substrate, FG5P (FG-G-G-G-G-P: FG, 6-deoxy-6-[(2-pyridyl)amino]-D-glucose residue; G, glucose residue; P, p-nitrophenyl residue; -, alpha-1,4-glycosidic bond). Chromatofocusing of urine from a healthy human was carried out. FG5P was digested with the fractions exhibiting alpha-amylase activity and each digest at an early stage was analyzed by HPLC. FG5P was hydrolyzed to FG3 (FG-G-G) and p-nitrophenyl alpha-maltoside (G-G-P), and to FG4 (FG-G-G-G) and p-nitrophenyl alpha-glucoside (G-P). The molar ratios of FG4 to FG3 (FG4/FG3) in the digests with basic fractions were larger than those in the digests of human pancreatic alpha-amylase (HPA, 1.11) and human salivary alpha-amylase (HSA, 0.51). Considering that the value for the AMY2B gene product with yeast (yHXA) is 1.88, a value of more than 1.11 implies that HXA exists. The amount of HXA was determined after removal of HSA on an anti-human salivary alpha-amylase antibody bound column. The FG4/FG3 values for six urine samples free from HSA were 1.23-1.26. Assuming that the FG4/FG3 value for HXA is the same as that for yHXA, the ratios of HXA and HPA were estimated to be 1:5.4-4.1. The results obtained showed that the AMY2B gene is usually expressed as HXA in healthy humans.