The human lactase persistence-associated SNP -13910*T enables in vivo functional persistence of lactase promoter-reporter transgene expression.

Lactase is the intestinal enzyme responsible for digestion of the milk sugar lactose. Lactase gene expression declines dramatically upon weaning in mammals and during early childhood in humans (lactase nonpersistence). In various ethnic groups, however, lactase persists in high levels throughout adulthood (lactase persistence). Genetic association studies have identified that lactase persistence in northern Europeans is strongly associated with a single nucleotide polymorphism (SNP) located 14 kb upstream of the lactase gene: -13910*C/T. To determine whether the -13910*T SNP can function in vivo to mediate lactase persistence, we generated transgenic mice harboring human DNA fragments with the -13910*T SNP or the ancestral -13910*C SNP cloned upstream of a 2-kb rat lactase gene promoter in a luciferase reporter construct. We previously reported that the 2-kb rat lactase promoter directs a post-weaning decline of luciferase transgene expression similar to that of the endogenous lactase gene. In the present study, the post-weaning decline directed by the rat lactase promoter is impeded by addition of the -13910*T SNP human DNA fragment, but not by addition of the -13910*C ancestral SNP fragment. Persistence of transgene expression associated with the -13910*T SNP represents the first in vivo data in support of a functional role for the -13910*T SNP in mediating the human lactase persistence phenotype.

Theodore E. Woodward Award: lactase persistence SNPs in African populations regulate promoter activity in intestinal cell culture.

Lactase-phlorizin hydrolase, lactase, is the intestinal enzyme responsible for the digestion of the milk sugar lactose. The majority of the world's human population experiences a decline in expression of the lactase gene by late childhood (lactase non-persistence). Individuals with lactase persistence, however, continue to express high levels of the lactase gene throughout adulthood. Lactase persistence is a heritable autosomal dominant condition and has been strongly correlated with several single nucleotide polymorphisms (SNPs) located ∼14 kb upstream of the lactase gene in different ethnic populations: -13910*T in Europeans and -13907*G, -13915*G, and -14010*C in several African populations. The coincidence of the four SNPs clustering within 100 bp strongly suggests that this region mediates the lactase non-persistence/persistence phenotype. Having previously characterized the European SNP, we aimed to determine whether the African SNPs similarly mediate a functional role in regulating the lactase promoter. Human intestinal Caco-2 cells were transfected with lactase SNP/promoter-reporter constructs and assayed for promoter activity. The -13907*G and -13915*G SNPs result in a significant enhancement of lactase promoter activity relative to the ancestral lactase non-persistence genotype. Such differential regulation by the SNPs is consistent with a causative role in the mechanism specifying the lactase persistence phenotype.

13915*G DNA polymorphism associated with lactase persistence in Africa interacts with Oct-1.

Lactase gene expression declines with aging (lactase non-persistence) in the majority of humans worldwide. Lactase persistence is a heritable autosomal dominant condition and has been strongly correlated with several single nucleotide polymorphisms (SNPs) located ~14-kb upstream (-13907, -13910 and -13915) of the lactase gene in different ethnic populations. In contrast to the -13907*G and -13910*T SNPs, the -13915*G SNP was previously believed not to interact with Oct-1. In the present study, however, Oct-1 is shown to interact with the -13915*G SNP region DNA sequence by EMSAs and gel supershift. In addition, Oct-1 is capable of enhancing promoter activity of a lactase promoter-reporter construct harboring the 13915*G SNP sequence in cell culture. Oct-1 binding to the -13907 to -13915 SNP region therefore remains a candidate interaction involved in lactase persistence.

Three forms of thermostable lactose-hydrolase from Thermus sp. IB-21: cloning, expression, and enzyme characterization.

Three thermostable lactose-hydrolases, namely, two beta-glycosidases (bglA and bglB) and one beta-galactosidase (bgaA) genes were cloned from the genomic library of Thermus sp. IB-21. The bglA, bglB, and bgaA consisted of 1311 bp (436 amino acid residues), 1296 bp (431 aa), and 1938 bp (645 aa) of nucleotides with predicted molecular masses of 49,066, 48,679, and 72,714 Da, respectively. These enzymes were overexpressed in Escherichia coli BL21(DE3) using pET21b(+) vector system. The recombinant enzymes were purified to homogeneity by a heat precipitation (70 degrees C, 40 min) and a Ni2+-affinity chromatography. The molecular masses of the purified enzymes estimated by SDS-PAGE agreed with their predicted values. All the purified enzymes showed their optimal pH at around 5.0-6.0. In contrast, the temperature profiles for activity and thermostability patterns were different for each enzyme. BglB beta-glycosidase displayed the best lactose hydrolysis activity of the three enzymes without substrate inhibition up to 200 mM lactose at 70 degrees C and pH 7.0. The specific activities (U/mg) of BglA, BglB, and BgaA on 138 mM lactose at 70 degrees C and pH 7.0 were 36.8, 160.3, and 8.5, respectively.

Effect of N-linked glycosylation on the aspartic proteinase porcine pepsin expressed from Pichia pastoris.

A study was undertaken to examine the effects of N-linked glycosylation on the structure-function of porcine pepsin. The N-linked motif was incorporated into four sites (two on the N-terminal domain and two on the C-terminal domain), and the recombinant protein expressed using Pichia pastoris. All four N-linked recombinants exhibited similar secondary and tertiary structure to nonglycosylated pepsin, that is, wild type. Similar K(m) values were observed, but catalytic efficiencies were approximately one-third for all mutants compared with the wild type; however, substrate specificity was not altered. Activation of pepsinogen to pepsin occurred between pH 1.0 to 4.0 for wild-type pepsin, whereas the glycosylated recombinants activated over a wider range, pH 1.0 to 6.0. Glycosylation on the C-terminal domain exhibited similar pH activity profiles to nonglycosylated pepsin, and glycosylation on the N-domain resulted in a change in activity profile. Overall, glycosylation on the C-domain led to a more global stabilization of the structure, which translated into enzymatic stability, whereas on the N-domain, an increase in structural stability had little effect on enzymatic stability. Finally, glycosylation on the flexible loop region also appeared to increase the overall structural stability of the protein compared with wild type. It is postulated that the presence of the carbohydrate residues added rigidity to the protein structure by reducing conformational mobility of the protein, thereby increasing the structural stability of the protein.

Soluble expression and purification of porcine pepsinogen from Pichia pastoris.

This paper presents a new system for the soluble expression and characterization of porcine pepsinogen from the methylotrophic yeast Pichia pastoris. The cDNA that encodes the zymogenic form of porcine pepsin (EC 3.4.23.1) was cloned into the EcoRI site of the vector pHIL-S1 downstream from the AOX1 alcohol oxidase promoter. After P. pastoris transformation, colonies were screened for expression of pepsinogen based on enzyme activity of the active form, pepsin. The recombinant enzyme was purified 138-fold by anion exchange and affinity column chromatography. Homogeneity was confirmed through SDS-PAGE, Western blot, and N-terminal sequencing. When compared to commercial pepsin, the recombinant pepsin had similar kinetic profiles, pH/temperature stability, and secondary/tertiary conformation. A glycosylated form was also isolated and found to exhibit kinetic and structural characteristics similar to those of the commercial and wild-type pepsin, but was slightly more thermal stable. The above results indicate that the P. pastoris expression system offers a convenient and efficient means to produce and purify a soluble form of pepsin(ogen).