A previously reported polymorphic plasma protein of dogs and horses, identified as apolipoprotein A-IV

By using immunoblotting with antiserum specific to human plasma apolipoprotein A-IV (apoA-IV), a previously reported polymorphic plasma protein of dogs viz postalbumin-2 (Pa2) and one of horses viz serum protein 2 (SP2), were identified as apoA-IV of these species. This along with earlier published results implied that: (1) both dog and horse show a high degree of polymorphism at the APOA4 locus with three common alleles in each of the two species; and (2) apoA-IV phenotyping in these two species can be done by analysing plasma/serum samples by a simple method of two-dimensional electrophoresis, conducted under non-denaturing conditions, followed by general-protein staining of gels.     

A previously reported polymorphic plasma protein of dogs and horses, identified as apolipoprotein A-IV

Summary. By using immunoblotting with antiserum specific to human plasma apolipoprotein A-IV (apoA-IV), a previously reported polymorphic plasma protein of dogs viz postalbumin-2 (Pa2) and one of horses viz serum protein 2 (SP2), were identified as apoA-IV of these species. This along with earlier published results implied that: (1) both dog and horse show a high degree of polymorphism at the APOA4 locus with three common alleles in each of the two species; and (2) apoA-IV phenotyping in these two species can be done by analysing plasma/serum samples by a simple method of two-dimensional electrophoresis, conducted under non-denaturing conditions, followed by general-protein staining of gels.     

Effects of variations in the APOA1/C3/A4/A5 gene cluster on different parameters of postprandial lipid metabolism in healthy young men

The APOA1/C3/A4/A5 gene cluster encodes important regulators of fasting lipids, but the majority of lipid metabolism takes place in the postprandial state and knowledge about gene regulation in this state is scarce. With the aim of characterizing possible regulators of lipid metabolism, we studied the effects of nine single nucleotide polymorphisms (SNPs) during postprandial lipid metabolism. Eighty-eight healthy young men were genotyped for APOA1 -2630 (rs613808), APOA1 -2803 (rs2727784), APOA1 -3012 (rs11216158), APOC3 -640 (rs2542052), APOC3 -2886 (rs2542051), APOC3 G34G (rs4520), APOA4 N147S (rs5104), APOA4 T29T (rs5092), and A4A5_inter (rs1263177) and were fed a saturated fatty acid-rich meal (1g fat/kg of weight with 60% fat, 15% protein and 25% carbohydrate). Serial blood samples were extracted for 11 h after the meal. Total cholesterol and fractions [HDL-cholesterol, LDL-cholesterol, trifacylglycerols (TGs) in plasma, TG-rich lipoproteins (TRLs) (large TRLs and small TRLs), apolipoprotein A-I and apolipoprotein B] were determined. APOA1 -2803 homozygotes for the minor allele and A4A5_inter carriers showed a limited degree of postprandial lipemia. Carriers of the rare alleles of APOA4 N147S and APOA4 T29T had lower APOA1 plasma concentration during this state. APOC3 -640 was associated with altered TG kinetics but not its magnitude. We have identified new associations between SNPs in the APOA1/C3/A4/A5 gene cluster and altered postprandial lipid metabolism.     

Haplotype analysis of the apolipoprotein gene cluster on human chromosome 11

Members of the apolipoprotein gene cluster (APOA1/C3/A4/A5) on human chromosome 11q23 play an important role in lipid metabolism. Polymorphisms in both APOA5 and APOC3 are strongly associated with plasma triglyceride concentrations. The close genomic locations of these two genes as well as their functional similarity have hindered efforts to define whether each gene independently influences human triglyceride concentrations. In this study, we examined the linkage disequilibrium and haplotype structure of 49 SNPs in a 150-kb region spanning the gene cluster. We identified a total of five common APOA5 haplotypes with a frequency of greater than 8% in samples of northern European origin. The APOA5 haplotype block did not extend past the 7 SNPs in the gene and was separated from the other apolipoprotein gene in the cluster by a region of significantly increased recombination. Furthermore, one previously identified triglyceride risk haplotype of APOA5 (APOA5*3) showed no association with three APOC3 SNPs previously associated with triglyceride concentrations, in contrast to the other risk haplotype (APOA5*2), which was associated with all three minor APOC3 SNP alleles. These results highlight the complex genetic relationship between APOA5 and APOC3 and support the notion that APOA5 represents an independent risk gene affecting plasma triglyceride concentrations in humans.     

The relationship among apolipoprotein(a) polymorphisms, the low-density lipoprotein receptor-related protein, and the very low density lipoprotein receptor genes, and plasma lipoprotein(A) concentration in the Czech population

Increased plasma concentration of lipoprotein(a) [Lp(a)] is an established independent risk factor for coronary artery disease (CAD), which is strongly genetically determined. This study was designed to investigate the relationship between the K-IV and (TTTTA)n apolipoprotein(a) [apo(a), protein; APOA, gene] polymorphisms, as well as the C766T low-density lipoprotein receptor-related protein (LRP) and the (CGG)n very low density lipoprotein receptor (VLDLR) polymorphisms on the one hand, and plasma Lp(a) levels in Czech subjects who underwent coronary angiography on the other hand. The lengths of the alleles of the APOA K-IV and (TTTTA)n polymorphisms were strongly inversely correlated with plasma Lp(a) levels in univariate analysis (r = -0.41, p < 10(-4) and r = -0.20, p < 0.01, respectively). Multivariate analysis revealed significant associations between the APOA polymorphisms studied and plasma Lp(a) levels in subjects expressing only one APOA K-IV allele (p < 10(-6) for K-IV and p < 0.001 for TTTTA). In subjects expressing both APOA K-IV alleles, the multivariate analysis revealed that only the APOA K-IV alleles were inversely correlated with plasma Lp(a) levels (p < 0.001). Associations between both the LRP and VLDLR gene polymorphisms and plasma Lp(a) levels were only of borderline significance (p < 0.06 and p < 0.07, respectively) and were not confirmed in multivariate analysis. In conclusion, both APOA length polymorphisms significantly influenced plasma Lp(a) concentration in the Czech population studied, and this circumstance could explain the association in this population observed earlier between APOA (TTTTA)n polymorphism and CAD (Benes et al. 2000). Only a minor role in the regulation of plasma Lp(a) levels is suggested for the C766T LRP and the (CGG)n VLDLR polymorphisms.     

Extended haplotypes and linkage disequilibrium between 11 markers at the APOA1-C3-A4 gene cluster on chromosome 11

One hundred fifty-four unrelated French Caucasian subjects were typed for 11 RFLPs at or near the APOA1-C3-A4 gene cluster on the long arm of chromosome 11. All subjects belonged to families having lived in the Toulouse area (in the southwest of France) for over three generations. Allele frequencies for each RFLP were in agreement with previous studies in Caucasian populations for the APOA1/SstI marker. Pairwise linkage disequilibrium was determined. Among the 55 pairs studied, 30 are newly reported. Only three significant nonrandom associations were found: APOA1/MspI-3'APOC3/SstI, APOA1/MspI-3'APOA4/XbaI, and APOA4/DraI-APOA4/XbaI. Extended 11-marker haplotypes were constructed. Haplotype frequencies were estimated by the maximum-likelihood procedure and compared to expected frequencies calculated under the assumption of equilibrium. Among the 37 estimated haplotypes, seven containing at least four nonrandomly associated alleles showed markedly increased frequencies. These results, obtained in a geographically homogeneous population, confirm the existence of disequilibrium in the apolipoprotein cluster, but to a lower extent than previously reported in Caucasian populations, which were geographically more heterogeneous.     

APOA1/C3/A4/A5基因簇的新载脂蛋白基因：APOA5及其研究进展

应用人和鼠的比较基因组学和功能基因组学方法,Pennaccio等和Vliet等分别在APOA1/C3/A4基因簇中发现新的载脂蛋白基因APOA5。人的APOA5基因编码366个氨基酸,与人APOA4、小鼠Apoa5高度同源。APOA5转基因小鼠其甘油三酯（TG）减少至野生型的1/3,而Apoa5基因敲除小鼠其TG却增加4倍。APOA5多态位点SNP3（-1131T>C）和S19W及单倍型APOA5*3有显著升高TG的作用。APOA5调节血浆TG水平的作用与APOC3作用相反,为冠心病等心血管疾病的易感因素。     

Blood protein polymorphisms in the donkey (Equus asinus)

Transferrin, albumin, 6-phosphogluconate dehydrogenase and vitamin D-binding protein polymorphisms were detected in 242 feral and domesticated Australian donkeys by polyacrylamide gel electrophoresis, starch gel electrophoresis, autoradiography, immunoblotting with specific antisera and activity staining. All four TF and two ALB variants were donkey specific while only one of the PGD variants was donkey specific. The two GC variants were electrophoretically identical to the Equus caballus F and S proteins. Available evidence suggested that the TF, ALB, PGD and GC systems are controlled by co-dominant alleles with frequencies of the most common alleles of each system being 0·831, 0·946, 0·957 and 0·861 respectively. Glucose phosphate isomerase and plasminogen were monomorphic in the Australian population of donkeys.     

Apolipoprotein A-IV genetic polymorphism and its impact on quantitative traits in normoglycemic and non-insulin-dependent diabetic Hispanics from the San Luis Valley, Colorado.

Apolipoprotein A-IV exhibits a common two-allele polymorphism in several human populations studied to date. Using isoelectric focusing and immunoblotting, we have analyzed plasmas from 188 non-insulin-dependent diabetic and 238 normoglycemic Hispanic individuals from the San Luis Valley, Colorado, to determine APOA4 genotype frequencies and to estimate the impact of the genotypes on quantitative traits. The frequencies of the two common alleles, APOA4*1 and APOA4*2, were 0.929 and 0.069, respectively, in normal subjects and 0.901 and 0.096, respectively, in diabetics. The third rare allele, APOA4*3, was detected sporadically in both groups. We studied the impact of APOA4 polymorphism on the levels of total plasma cholesterol, HDL cholesterol and its subfractions (HDL3 and HDL2), LDL cholesterol, triglycerides, glucose, and insulin. We observed no significant effect of the APOA4 polymorphism on any trait in diabetics. However, we did note a significant sex-specific effect in normoglycemic females on the level of total HDL cholesterol (p = 0.001) and its subfractions HDL2 (p = 0.043) and HDL3 (p = 0.001). The effect of the APOA4*2 allele in normal Hispanic females was to lower the total HDL, HDL2, and HDL3 cholesterol by 8.75 mg/dl, 2.37 mg/dl, and 5.36 mg/dl, respectively, compared to the common APOA4*1 allele.     

Discovery of Tumor Markers for Gastric Cancer by Proteomics

Gastric cancer (GC) has a high rate of morbidity and mortality among various cancers worldwide. The development of noninvasive diagnostic methods or technologies for tracking the occurrence of GC is urgent, and searching reliable biomarkers is considered.This study intended to directly discover differential biomarkers from GC tissues by two-dimension-differential gel electrophoresis (2D-DIGE), and further validate protein expression by western blotting (WB) and immunohistochemistry (IHC).Pairs of GC tissues (gastric cancer tissues and the adjacent normal tissues) obtained from surgery was investigated for 2D-DIEG.Five proteins wereconfirmed by WB and IHC, including glucose-regulated protein 78 (GRP78), glutathione s-transferase pi (GSTpi), apolipoprotein AI (ApoAI), alpha-1 antitrypsin (A1AT) and gastrokine-1 (GKN-1). Among the results, GRP78, GSTpi and A1ATwere significantlyup-regulated and down-regulated respectively in gastric cancer patients. Moreover, GRP78 and ApoAI were correlated with A1AT for protein expressions.This study presumes these proteins could be candidates of reliable biomarkers for gastric cancer.     

Rare deficiency types of alpha 1-antitrypsin: electrophoretic variation and DNA haplotypes.

A deficiency of the plasma protease inhibitor alpha 1-antitrypsin (alpha 1AT), is usually associated with the deficiency allele PI*Z. However, other alleles can also produce a deficiency. Some of these rare deficiency alleles produce a low concentration (3%-15% of normal) of alpha 1AT and include Mmalton, Mduarte, Mheerlen, and Mprocida. Null, or nonproducing, alleles are associated with trace amounts (less than 1%) of plasma alpha 1AT. We have identified, using isoelectric focusing, the deficiency alleles in 222 patients (68 children and 154 adults) with alpha 1AT deficiency. In addition to PI*Z, we found low-producing alleles PI*Mmalton and PI*Mcobalt and four null (PI*QO) alleles. On the basis of a population frequency of .0122 for PI*Z, frequencies for other deficiency alleles are 1.1 x 10(-4) for PI*Mmalton, 2.5 x 10(-5) for PI*Mcobalt (which may be the same as that for PI*Mduarte, and 1.4 x 10(-4) for all null alleles combined. Using 12 polymorphic restriction sites with seven different restriction enzymes, we have obtained DNA haplotypes for each of the rare deficiency types. All of the rare deficiency alleles can be distinguished from PI*Z by their DNA haplotype, and most can be distinguished from each other. DNA haplotypes are useful to indicate the presence of new types of null alleles, to identify genetic compounds for rare deficiency alleles, and to identify the original normal allele from which each deficiency allele is derived.     

Genetic linkage analysis between protein polymorphisms and the FecB major gene in sheep

A genetically linked marker locus is sought for the Booroola gene (FecB), a major gene which confers increased prolificacy in sheep. We examined 18 polymorphic proteins in sheep and found 10 to be informative in half-sib families where the Booroola gene was segregating. Recombination was observed between each of the protein loci and the Booroola gene. The loci and exclusion distance for each (calculated as the recombination fraction where the lod score was equal to -2.0) are as follows: NADH diaphorase, DIA1 (9.2 cM); arylesterase, EsA (11.9 cM); haemoglobin beta chain, HBB (17.5 cM); leucine amino peptidase, LAP (19.7 cM); malic enzyme, ME1 (14.8 cM); ovine plasminogen antigen, OPA (12.6 cM); alpha-1-protease inhibitor, PI2 (5.7 cM), erythrocyte 'X' protein, Prot-X (25.3 cM); post transferrin, PTF (2.2 cM); transferrin, TF (33.8 cM).     

Genetic polymorphism of plasma α1B-glycoprotein and transferrin in arctic and silver foxes

Plasma samples of 235 foxes from 38 complete families (14 of arctic foxes, 21 of silver foxes and 3 with arctic x silver fox hybrid offspring) were analysed by one-dimensional horizontal polyacrylamide gel electrophoresis (PAGE) pH 9.0 followed by general-protein staining of gels. A major postalbumin of fox plasma was identified as alpha 1B-glycoprotein (alpha 1B) by using immunoblotting with antiser m specific to human or pig plasma alpha 1B. Four codominant, autosomal alleles of alpha 1B were found in arctic foxes. Two transferrin (TF) alleles (TfF, TfS) were observed in arctic foxes and two (TfD, Tff) in silver foxes; the TF F type of both of the fox species showed identical electrophoretic mobilities. The arctic foxes showed a high degree of polymorphism for both TF and alpha 1B. The silver foxes showed a scarce polymorphism of TF and were monomorphic for alpha 1B. The arctic fox, silver fox and their hybrids could be clearly differentiated from one another by their plasma protein patterns obtained by the PAGE method.     

Human CRISP-3 binds serum α1B-glycoprotein across species

Background

CRISP-3 was previously shown to be bound to α₁B-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular such that involve cell cultures, binding proteins present in sera might interfere in the experiments.

Methods

We examined sera from five different animal species for CRISP-3 binding proteins using gel filtration and ligand blotting. We developed a rapid method for isolation of proteins that bind to human CRISP-3 and identified the isolated proteins by mass spectrometry and N-terminal sequencing.

Results

We identified A1BG as a CRISP-3 binding protein in sera from cow, horse and rabbit. CRISP-3 bound kininogen 1 in mouse serum, whereas rat serum showed no CRISP-3 binding activity. In equine serum, we furthermore detected a possible CRISP, already bound to A1BG.

General significance

It seems to be a common mechanism that A1BGs bind CRISPs, also across species. Apart from the possible physiological implications hereof, complex binding of CRISPs by A1BG (and other proteins) may interfere with the detection and function of CRISPs, when these are studied in the presence of animal sera.     

Cysteine-rich secretory protein 3 is a ligand of alpha1B-glycoprotein in human plasma

Human cysteine-rich secretory protein 3 (CRISP-3; also known as SGP28) belongs to a family of closely related proteins found in mammals and reptiles. Some mammalian CRISPs are known to be involved in the process of reproduction, whereas some of the CRISPs from reptiles are neurotoxin-like substances found in lizard saliva or snake venom. Human CRISP-3 is present in exocrine secretions and in secretory granules of neutrophilic granulocytes and is believed to play a role in innate immunity. On the basis of the relatively high content of CRISP-3 in human plasma and the small size of the protein (28 kDa), we hypothesized that CRISP-3 in plasma was bound to another component. This was supported by size-exclusion chromatography and immunoprecipitation of plasma proteins. The binding partner was identified by mass spectrometry as alpha(1)B-glycoprotein (A1BG), which is a known plasma protein of unknown function and a member of the immunoglobulin superfamily. We demonstrate that CRISP-3 is a specific and high-affinity ligand of A1BG with a dissociation constant in the nanomolar range as evidenced by surface plasmon resonance. The A1BG-CRISP-3 complex is noncovalent with a 1:1 stoichiometry and is held together by strong electrostatic forces. Similar complexes have been described between toxins from snake venom and A1BG-like plasma proteins from opossum species. In these cases, complex formation inhibits the toxic effect of snake venom metalloproteinases or myotoxins and protects the animal from envenomation. We suggest that the A1BG-CRISP-3 complex displays a similar function in protecting the circulation from a potentially harmful effect of free CRISP-3.     

Contribution of APOA5 gene variants to plasma triglyceride determination and to the response to both fat and glucose tolerance challenges

The aim of this study was to investigate the influence of APOA5 variants on fasting lipids and to the response to both an oral fat tolerance test (OFTT) and an oral glucose tolerance test (OGTT). The association of two APOA5 SNPs [S19W (SNP5), -1131T>C (SNP3)] and an APOA4/A5 intergenic SNP [-12238T>C (SNP4)] were examined in healthy young men (n=774) who had undergone both an OFTT and an OGTT. Both -1131T>C and S19W rare alleles were associated with triglyceride (TG)-raising effects (11%, P=0.008; 21% (in cases), P<0.026, respectively) and showed additive effects on TG. None of the variants influenced the responsiveness to the OFTT after correcting for baseline TG. Homozygosity for the -12238T>C rare allele was associated with higher waist to hip ratio (P<0.0006), systolic blood pressure (P=0.012) and AUC and peak of insulin after OGTT (P=0.003 and P=0.027, respectively), traits that define the metabolic syndrome. Our results strongly support the role of APOA5 in determining plasma TG levels in an age-independent manner and highlight the importance of the APOC3/A4/A5 gene cluster in both TG and metabolic homeostasis.     

Twenty-seven protein polymorphisms by two-dimensional electrophoresis of serum, erythrocytes, and fibroblasts in two pedigrees

Twenty-seven independent polymorphic loci were detected by two-dimensional electrophoresis (2DE) of serum, erythrocytes, and fibroblasts in two large families and analyzed for linkage to classical genetic markers. We detected seven serum, four erythrocyte, and 17 fibroblast protein loci that exhibited charge variation in these two families and in a sample of unrelated individuals. The genetic basis of protein variants was confirmed by quantitative gene-dosage dependence and by conformance to Mendelian transmission in the two families, except for four rare variants for which transmission analysis was not possible. Linkage analysis demonstrated that each of the variants represent products of independent loci, with the exception of erythrocyte locus (RBC4), which we also detected in fibroblasts (NC27). Two allozyme polymorphisms, glyoxalase-1 (GLO1) and phosphoglucomutase-3 (PGM3) were specifically identified here based on genotypic concordance and molecular mass. Unknown fibroblast protein (NC22) may be linked to apolipoprotein E (lod score = 2.8 at theta m = theta f = 0), while a serum protein locus (SER1) may be linked to alpha-haptoglobin (lod score = 2.54 at theta m = .20, theta f = .01). Six of seven polymorphic serum loci were previously located on two-dimensional gels: alpha-1 antitrypsin (PI), Gc-globulin (GC), alpha-2 HS glycoprotein (HSGA), alpha-haptoglobin (HP), and two apolipoproteins (APOE and APOA4). Six of 17 polymorphisms detected in fibroblasts were positionally identical to polymorphic loci seen in lymphocytes. These studies indicate a minimum level of average protein charge heterozygosity of approximately 2.2% for the most predominant human cellular proteins and of 5.6% for the most predominant proteins of serum.     

Genetic structure of a Sakha population from Siberia and ethnic affinities

The red cell enzymes ACP1, ESD, GLO1, PGM1 and RDS and the serum proteins GC, HP, PI, and TF were determined for samples of 150 and 144 Sakha, respectively. The Sakha, a Turkic-speaking population, inhabit the Sakha-Yakutia Republic in northeastern Siberia. High gene frequencies were found for ACP1*A, GLO1*1 and GC*1F, whereas no P1*S or P1*Z alleles were found. In addition, 1 heterozygous phenotype with ACP1*C and 2 heterozygous phenotypes with ESD*7 were found. The genetic distance measures show close affinities of the Sakha population to Buryats (especially Western Buryats), Mongols, and Evenks, whereas the genetic distance to Turkic-speaking Altay and Tuvan populations is great.     

3'' -> 5'' Exonucleases of DNA Polymerases ε and δ Correct Base Analog Induced DNA Replication Errors on opposite DNA Strands in Saccharomyces Cerevisiae

The base analog 6-N-hydroxylaminopurine (HAP) induces bidirectional GC -> AT and AT -> GC transitions that are enhanced in DNA polymerase ε and δ 3' -> 5' exonuclease-deficient yeast mutants, pol2-4 and pol3-01, respectively. We have constructed a set of isogenic strains to determine whether the DNA polymerases δ and ε contribute equally to proofreading of replication errors provoked by HAP during leading and lagging strand DNA synthesis. Site-specific GC -> AT and AT -> GC transitions in a Pol(+), pol2-4 or pol3-01 genetic background were scored as reversions of ura3 missense alleles. At each site, reversion was increased in only one proofreading-deficient mutant, either pol2-4 or pol3-01, depending on the DNA strand in which HAP incorporation presumably occurred. Measurement of the HAP-induced reversion frequency of the ura3 alleles placed into chromosome III near to the defined active replication origin ARS306 in two orientations indicated that DNA polymerases ε and δ correct HAP-induced DNA replication errors on opposite DNA strands.