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PubMed1992

Biological characterization of the isoforms of urinary human follicle-stimulating hormone contained in a purified commercial preparation.

The main physicochemical and biological properties of the several isoforms of urinary follicle-stimulating hormone (uFSH) present in a commercially available uFSH preparation were analysed. Purified urinary FSH was submitted to chromatofocusing and several immunoactive forms of uFSH with isoelectric points (pI) ranging from 5.5 to 3.8 were identified. An additional isoform was detected after passing through the chromatofocusing column a 1.0 M NaCl solution (salt peak). Each uFSH isoform or pool of neighbouring isoforms (pI value 5.5-5.1, pool I, 3.8 +/- 1.0% of total immunoactivity recovered; pI value 5.0-4.6, pool II, 18.4 +/- 3.6% of total; pI value 4.5-4.3, pool III, 14.9 +/- 1.5% of total; pI value 4.1, pool IV, 8.2 +/- 1.4% of total; salt peak, pool V, 51.1 +/- 6.4% of total) eluted as single FSH peaks after Sephadex G-100 exclusion chromatography (apparent M(r) 60,000). Even though FSH present within each pool was recognized by a receptor preparation, the receptor binding activity expressed as the radioreceptor assay/radioimmunoassay (RRA/RIA) activity ratio varied with the pI value of the particular uFSH isoform tested; starting from a pI value of 5.5, the receptor binding activity of FSH decreased from 5.9 +/- 0.39 to 2.4 +/- 0.19, as the pI value of the corresponding isoform declined. A similar trend was observed when the potency of each isoform was assessed by an in-vitro bioassay.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1997

The dual functions of Tetrahymena citrate synthase are due to the polymorphism of its isoforms.

Previous studies have shown that Tetrahymena citrate synthase and the Tetrahymena 14-nm filament protein are encoded by a single gene and translated from one species of mRNA, and that they are identical in terms of molecular weight, antigenicity, and some enzymatic properties. In this study, using two-dimensional gel electrophoresis, we demonstrated that the citrate synthase comprised pI 7.7 and 8.0 isoforms, while the 14-nm filament protein comprised three isoforms with isoelectric points of 7.7, 8.0, and 8.4. The amino acid sequences of the NH2-terminal portions of all isoforms were identical and the peptide maps with V8 protease were almost the same. In addition, when the citrate synthase activity of each isoform was measured after separation by non-urea isoelectric focusing without denaturing treatment, the pI 7.7 and/or pI 8.0 isoforms exhibited the citrate synthase activity, but the pI 8.4 isoform only found for the 14-nm filament protein did not possess this activity. These results suggest that the polymorphism of these isoforms is caused by some posttranslational modifications, and that it may have resulted in the different compartmentalization and functions of Tetrahymena citrate synthase and the 14-nm filament protein.

Amino Acid Sequence↗
PubMed1998

Cloning and characterization of three isoforms of OS-9 cDNA and expression of the OS-9 gene in various human tumor cell lines.

OS-9 gene is frequently coamplified with CDK4 gene in human sarcomas. We isolated and characterized three isoforms of OS-9 cDNA found in a myeloid leukemia HL-60 cDNA library. Isoform 1 consisted of 2,700 bp, from which a 667 amino acid sequence was deduced and found to be identical with that of OS-9 cDNA from osteosarcoma cells [Su et al. (1996) Mol. Carcinogen. 15, 270-275]. Isoform 2 cDNA lacked a 165 nucleotide sequence in the coding region. Isoform 3 cDNA had an additional 45 bp deletion in the coding region. Isoforms 2 and 3 encode 612 and 597 amino acid polypeptides, respectively. Comparison of their cDNA sequences with the genomic structure indicated that three isoforms are splice variants. Reverse transcription-polymerase chain reaction analysis showed predominant expression of isoform 2 mRNA in myeloid leukemia HL-60 cells, osteosarcoma OsA-CL cells and rhabdomyosarcoma Rh30 cells. Northern blotting revealed similar levels of expression of OS-9 gene in various tumor cell lines of sarcoma cells, carcinoma cells and myeloid leukemia cells, but 3-4 times higher expression in OsA-CL cells and Rh30 cells containing a homogeneously staining region of 12q13-15. OS-9 expression decreased in differentiation-induced HL-60 cells. Possible involvement of the OS-9 gene in cell growth is discussed.

Alternative Splicing↗
PubMed1991

Identification and distribution of tropomyosin isoforms in chicken digestive canal.

Two-dimensional gel electrophoresis of eight digestive organ extracts from 60-day-old chicks was performed. Judging from the similarity of their protein maps, the organs were classified into the following four types: 1) esophagus type, 2) proventriculus type, 3) gizzard type, and 4) intestine type. In four representative organs of these types, the distribution of tropomyosin isoforms was examined, and four high- and five low-Mr-type isoforms in addition to alpha and beta isoforms were detected in the embryonic organs. In the adult organs, however, there were three high- and four low-Mr-type isoforms, which were restricted to the mucous membrane, in addition to alpha and beta isoforms. Immunoblot analysis indicated that the high- and low-Mr-type isoforms in the embryo corresponded with those in the adult mucous membrane, but differences in the number and amount of the isoforms were found between the embryo and the adult mucous membrane.

Animals↗
PubMed1993

Cloning of the rat aortic smooth muscle Na+/Ca2+ exchanger and tissue-specific expression of isoforms.

The amino acid sequences of two isoforms of the rat aortic smooth muscle Na+/Ca2+ exchanger have been deduced by cloning and sequencing the cDNAs. These isoforms are identical in nucleotide sequence except that one has a 23-amino acid insertion at amino acid position 570. They are highly homologous to the canine cardiac exchanger except for the NH2-terminal portion and part of the large central hydrophilic domain (amino acid residues 570-631). They are 902 and 925 (with the insertion) amino acid long with calculated molecular masses of 100,676 and 103,200 (with the insertion), respectively, if the NH2-terminal 32-amino acid residues are eliminated as a cleaved signal sequence. Amplification of the variable region (amino acids 570-631) of the exchanger by means of the reverse transcriptase-polymerase chain reaction and DNA sequencing revealed that many isoforms of the exchanger are expressed in different rat tissues. The two clones isolated in this study are the predominant isoforms expressed in aorta, stomach, liver, and kidney. In cardiac and skeletal muscles, another isoform is dominant, which is equivalent to the canine cardiac exchanger. In brain, a third type is predominantly expressed. Alignment of the nucleotide sequences of these isoforms and Southern blot analysis of rat genomic DNA suggested that each isoform is generated through alternative splicing of the primary transcript.

Alternative Splicing↗
PubMed2001

Gene duplication events producing muscle (M) and brain (B) isoforms of cytoplasmic creatine kinase: cDNA and deduced amino acid sequences from two lower chordates.

Creatine kinase (CK) is coded for by at least four loci in higher vertebrates--two cytoplasmic isoforms, muscle (M) and brain (B), and two mitochondrial isoforms, sarcomeric and ubiquitous. M is expressed primarily in skeletal muscle, while B is expressed in a variety of cells, including cardiac and smooth muscle fibers, neurons, transport epithelia, and photoreceptors. M and B subunits form very stable homodimers (MM [M-CK], BB [B-CK]) and heterodimers (MB). M-CK is capable of binding to the M line of the myofibril, thereby creating an energy transfer microcompartment; BB and MB CKs are not. M- and B-like CKs are present in all vertebrates yet examined, including fish. Cytoplasmic, dimeric CKs are widely distributed in the invertebrates. The only available amino acid sequence for an invertebrate dimeric CK, that of the protostome polychaete Chaetopterus variopedatus, is just as similar to the vertebrate M isoform as to the B isoform. Echinoderms lack dimeric, cytoplasmic CKs, which appear to be replaced by a dimeric arginine kinase which evolved secondarily from CK. Thus, it is likely that the gene duplication event producing the M and B isoforms occurred after the divergence of the chordates from echinoderms. To narrow down the timing of this duplication event, we obtained the cDNA and deduced amino acid sequences of dimeric CKs from the tunicate Ciona intestinalis (subphylum Urochordata) and the lancelet Branchiostoma floridae (subphylum Cephalochordata). Our results show that these CKs are strikingly similar to both invertebrate and vertebrate CKs. However, phylogenetic analyses by neighbor-joining and parsimony show that these two enzymes appeared to have diverged before the point of divergence of the M and B isoforms. Thus, the gene duplication event for formation of the muscle and brain isoforms of CK most likely occurred during the radiation of the fish, a time noted for gene duplication events at a variety of other loci.

Amino Acid Sequence↗
PubMed1999

Effects of acute exposure to PCBs 126 and 153 on anterior pituitary and thyroid hormones and FSH isoforms in adult Sprague Dawley male rats.

3,3'4,4',5-Pentachlorobiphenyl (PCB 126) and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) were administered to adult male rats in order to identify sensitive indicators of endocrine disruption. We tested the hypothesis that PCB exposure modifies follicle-stimulating hormone (FSH) pituitary isoforms, as well as the pituitary and serum concentrations of FSH, luteinizing hormone (LH), growth hormone, prolactin, and thyroid-stimulating hormone (TSH). Effects on serum levels of thyroxine (T4) and testosterone (T), and prostate androgen receptor content, were also tested. In one experiment, 5 groups of 8 rats each received two i.p. injections, one day apart, of either corn oil or 6.25, 25, 100 or 400 micrograms/kg/day of PCB 126. Decreases (p < 0.05) in the serum concentrations of T4 and LH started at doses of 25 and 100 micrograms/kg/day, respectively. Serum FSH concentrations were reduced (p = 0.07) in the highest dose group. In contrast, pituitary content of FSH and LH increased with PCB-126 doses (p = 0.004, p = 0.002, respectively). Despite changes in reproductive hormones, PCB-126 had no effect on the androgen receptor content of the prostate. The effect of PCB-126 was tested in the hemicastrated rat, and suggested adverse effects on testosterone secretion. To test the effects of PCB exposure on FSH pituitary isoforms, 4 groups of 10 male rats received two i.p. injections, one day apart, of either corn oil, PCB 153 (25 mg/kg/day), estradiol-17 beta (E2; 20 micrograms/kg/day), or PCB 126 (0.1 mg/kg/day). Serum T4 levels were higher (p < 0.01) in the E2 and PCB 153 groups, and slightly reduced in the PCB 126-treated groups, compared to controls. Simultaneous purification of pituitary FSH and TSH isoforms was performed by HPLC, using two chromatofocusing columns in series. In contrast to TSH isoforms, the distribution of FSH isoforms over the chromatography run differed slightly between treatment groups; the amounts of FSH isoform eluted during the pH gradient were lower (p < 0.05) in E2 and PCB 153-treated rats than in control or PCB 126-treated rats. The similarity between the effects of E2 and PCB 153 on T4 and FSH isoforms supports the contention that PCB 153 possesses estrogenic properties. Serum LH and T4 concentrations were the most sensitive and practical endocrine indicators of PCBs 126 and 153 exposure in male rats.

Animals↗
PubMed2026

k-mer-based Upstream Preprocessing of long reads for Isoform Discovery.

Eukaryotic genes can encode multiple protein isoforms based on alternative splicing of their transcribed regions. Most modern novel isoform discovery methods function by identifying and assembling exon splice junctions from an RNA-seq sample. However, splice junctions can only be accurately annotated with time-intensive dynamic programming alignment. This manuscript introduces KuPID, a method for preprocessing long RNA-seq reads with the goal of better identifying novel isoform transcripts. KuPID utilizes k-mer sketching as a prefilter to quickly pseudo-align reads to known reference isoforms. Full alignment need only then be applied to reads that are most relevant to isoform discovery. Not only does KuPID speed up the discovery pipeline, it also increases downstream accuracy by filtering out extraneous reads. KuPID preprocessing simultaneously increases the f1 accuracy of isoform discovery pipelines by up to 11.6 points while decreasing the runtime by a factor of 2-3&#xd7;;. An optional mode permits a KuPID sample to be paired with both isoform discovery and transcript quantification.

Journal Article↗
PubMed1993

Molecular heterogeneity of photosystem I. psaD, psaE, psaF, psaH, and psaL are all present in isoforms in Nicotiana spp.

The protein composition of photosystem I (PSI) was examined in Nicotiana spp. by high-resolution polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and immunoblot analysis. Five PSI proteins show polymorphism in an amphidiploid species, Nicotiana tabacum, but not in its ancestral diploid species, Nicotiana sylvestris and Nicotiana tomentosiformis. These Nicotiana spp. appear to have at least 18 PSI proteins per genome that range in molecular mass from 3 to 20 kD. They include the products of nuclear genes psaD, psaE, psaF, psaG, psaH, psaK, and psaL, the product of chloroplast gene psaC, N-terminally blocked proteins of 4.5 and 3.0 kD, and an unidentified protein of 12.5 kD. The psaD, psaF, psaH, and psaL products have two isoforms each that are distinguished by different mobilities in polyacrylamide gel electrophoresis, and the psaE product has four isoforms. The two isoforms of the psaD product have distinct amino acid sequences, indicating that they are encoded by different genes within the genome. Four isoforms of the psaE products can be classified into two groups by N-terminal amino acid sequence, indicating that at least two psaE genes are present in the genome. To examine whether the polymorphic nature of PSI is peculiar to Nicotiana spp., we carried out immunoblot analysis of the psaD and psaE products in isogenic lines of tomato (Lycopersicon esculentum), Arabidopsis thaliana, red bean (Vigna angularis), and corn (Zea mays). Two electrophoretically distinct isoforms were found for the psaD products of tomato, A. thaliana, and corn, and two isoforms of psaE products were detected in tomato, A. thaliana, and red bean. These results suggest that the nuclear-encoded subunits of PSI, except for the psaG and psaK products, generally have two isoforms.

Amino Acid Sequence↗
PubMed1992

alpha-Amylase Isoforms are Posttranslationally Modified in the Endomembrane System of the Barley Aleurone Layer.

The subcellular site of the posttranslational modification of alpha-amylase was investigated in aleurone layers of barley (Hordeum vulgare L. cv Himalaya). Aleurone layers of Himalaya barley synthesize and secrete two groups of alpha-amylase isoforms, referred to as low-isoelectric point (low-pl) or HAMY1 and high-pl or HAMY2, when incubated in gibberellic acid and CaCl(2). Whereas homogenates of aleurone layers contain four isoforms of HAMY1 with pls 4.90, 4.72, 4.64, and 4.56, incubation media contain predominantly isoforms 4.72 and 4.56. Microsomal membranes isolated from aleurone layers contain all four isoforms of HAMY1. Microsomal membranes can be resolved into two peaks by isopycnic density gradient centrifugation: a peak of heavy membranes with endoplasmic reticulum and Golgi apparatus (GApp) marker enzyme activities and a peak of light membranes with characteristics of the GApp. The heavy membranes contain proportionally more HAMY1 pl 4.90 and 4.64 isoforms, whereas light membranes contain a higher proportion of pl 4.72 and 4.56 isoforms. Experiments with the ionophore monensin show that membranes of the GApp as well as the endoplasmic reticulum are involved in the posttranslational modification of HAMY1 isoforms. Monensin inhibits the secretion of alpha-amylase and causes the enzyme to accumulate within the cell. Precursor forms of HAMY1 accumulate in light membranes isolated from monensin-treated aleurone layers indicating that the GApp is involved in the conversion of the precursor to the secreted forms of the enzyme.

Journal Article↗
PubMed1998

Phorbol ester-induced G1 arrest in BALB/MK-2 mouse keratinocytes is mediated by delta and eta isoforms of protein kinase C.

We investigated the possible negative regulation of the cell cycle by protein kinase C (PKC) isoforms in synchronously grown BALB/MK-2 mouse keratinocytes, in which PKC isoforms were overexpressed by using the adenovirus vector Ax. Cells at the G1/S boundary of the cell cycle were the most sensitive to the inhibitory effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC agonist, resulting in G1 arrest. TPA-induced inhibition of DNA synthesis was augmented by overexpression of the eta and delta isoforms, but rescued by the dominant-negative and antisense eta isoforms. In contrast, the alpha and zeta isoforms showed no effect on DNA synthesis with or without TPA treatment. Immunoblotting indicated cell cycle-dependent expression of the eta isoform, being highest in cells at the G1/S boundary. The present study provides evidence that the eta and delta isoforms of PKC are involved in negative regulation of cell cycle at the G1/S boundary in mouse keratinocytes.

Adenoviridae↗
PubMed1994

17 beta-oestradiol counteracts the formation of the more acidic isoforms of follicle-stimulating hormone and luteinizing hormone after menopause.

OBJECTIVE: When the gonadotrophin levels increase at midcycle, more basic isoforms of FSH and LH appear in the circulation. However, when these gonadotrophins increase at menopause more acidic forms appear. The present study was done to see whether chronic 17 beta-oestradiol (E2) administration to post-menopausal women could counteract the formation of the more acidic isoforms after the menopause. DESIGN: Serum samples were obtained from 16 post-menopausal women, mean age 70 years (range 63-84 years), 46-169 days after the subcutaneous insertion of a 20-mg E2-implant. FSH, LH and E2 in the sera were measured with fluoroimmunoassays. The median charge and the degree of charge heterogeneity of the FSH and LH isoforms were determined for each serum by electrophoresis in 0.1% agarose suspension. Sera from an age-matched control group were analysed in parallel. RESULTS: The E2 levels in the E2-treated women were 230-570 pmol/l, within the range expected during the mid-luteal phase of the normal menstrual cycle. The mean serum FSH and LH levels were similar to normal follicular phase FSH and LH levels (8.6 and 20.8% respectively of the control group). It was estimated that individual serum specimens from both groups contained 20-30 different isoforms for both FSH and LH. The median charges of the isoforms of FSH and LH were more basic in all the E2-treated subjects than in their corresponding untreated controls. The mean median charge for FSH was close to the values for the follicular and luteal phases and that for LH close to that for the luteal phase. In some E2-treated women the isoforms were even more basic with a charge similar to that at the midcycle peak. The degree of charge heterogeneity for the E2-treated group was significantly (P < 0.001) larger than for the controls and similar to that during the normal menstrual cycle. CONCLUSION: Chronic E2 administration to post-menopausal women counteracted the formation of more acidic isoforms of both FSH and LH after the menopause.

Aged↗
PubMed1994

Analysis of Oct2-isoform expression in lipopolysaccharide-stimulated B lymphocytes.

Oct2-isoform expression in splenic B cells stimulated with lipopolysaccharide or lipopolysaccharide plus phorbol-di-butyrate was analysed by cDNA cloning. The frequency of Oct2-positive clones was 1/15,000 in both libraries. Two new isoforms were found that generate novel amino- or carboxy-terminal sequences. An isoform lacking exon 11 destroyed the carboxy-terminal leucin-zipper region and introduced a frame shift creating a novel, proline-rich carboxy terminus. A new exon containing a highly basic region (4c) was characterized, between exons 4 and 5. This exon was inserted between glutamine-rich regions 2 and 3, carboxy terminal of a tentative leucine-zipper structure. In addition, a new combination isoform containing Oct2a's amino terminal insert (exon 7a) and Oct2b's carboxy terminal insert (exon 13) was found that created a novel large isoform, Oct2ab. More frequent use of the classical Oct2a and Oct2b isoforms was observed in the lipopolysaccharide-stimulated B cells, while a preference for the Oct2ab and Oct2ba isoforms was observed in lipopolysaccharide plus phorbol-di-butyrate-treated cells.

Amino Acid Sequence↗
PubMed1997

Myeloperoxidase isoform activities released by human neutrophils in response to dental and periodontal bacteria.

Myeloperoxidase is an enzyme released by polymorphonuclear leukocytes which has been used, experimentally, as an indicator of periodontal disease activity when measured in gingival crevicular fluid. There are three myeloperoxidase isoforms: MPO I, MPO II and MPO III. We examined the activities of myeloperoxidase isoforms released by neutrophils in response to serum-opsonized Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Eikenella corrodens, Capnocytophaga sputigena and Streptococcus sanguis. Isoform activities were determined using intermediate-pressure liquid chromatography and microenzyme assay. A. actinomycetemcomitans stimulated higher levels of myeloperoxidase release than any other oral bacteria unless pre-opsonized with serum (or protein-A-purified immunoglobulin) from an individual with localized juvenile periodontitis. Most oral bacteria stimulated the release of all myeloperoxidase isoforms with a profile enriched in MPO I and diminished in MPO III. Exceptionally, serum-opsonized A. actinomycetemcomitans stimulated myeloperoxidase isoform release in proportion to the neutrophil granule constituency with or without localized juvenile periodontitis serum pre-opsonization. Because myeloperoxidase isoform profiles reflect how neutrophils were stimulated, isoform analysis may refine future diagnostic tests based upon myeloperoxidase.

Adult↗
PubMed1991

Regulation of the expression of the sex-specific isoforms of cytochrome P-450 in rat liver.

The hepatic metabolism of steroid hormones and of xenobiotics frequently depends on the expression of the sex-specific isoforms of cytochrome P-450 and on differences in sex hormones. Following biochemical, immunological and molecular biological investigations, it was shown that in adult rat liver there exist at least four male-specific and one female-specific isoforms of cytochrome P-450. The designation of these sex-specific genes is IIC11, IIIA2, IIC13 and IIA2 in males, and IIC12 in females. The irreversible programming of the expression of these isoforms of cytochrome P-450 in adulthood occurs during the perinatal period of life, and is named enzyme imprinting. One of the main factors that regulates the expression of the sex-specific isoforms of cytochrome P-450 is the level of androgens in the blood. Castration of adult rats decreased the level of the male isoforms of cytochrome P-450 and the activity of the monooxygenase enzyme system that remained higher than in intact females. The mechanism of enzyme imprinting can be explained as follows: neonatal androgens program the secretion of hypothalamic hormones, somatostatin and growth-hormone-releasing factor. These factors determine the type of growth hormone secretion in adult rats, and this controls the type of sex-specific isoforms of cytochrome P-450 expressed in adulthood. Metabolic regulation similar to that outlined above was shown to occur for several metabolism-dependent chemical carcinogens. Such a pathway may explain the different sensitivity displayed by male and female rats to treatment with these carcinogenic agents. One possible way of modulating the expression of some isoforms of cytochrome P-450 in adult rats is by treating neonates with specific xenobiotics that change the constitutive expression of neonatal androgens. It appears that this enzyme imprinting plays an important role in determining the individual sensitivity to the carcinogenic effects of chemicals.

Animals↗
PubMed1993

Comparison of crotoxin isoforms reveals that stability of the complex plays a major role in its pharmacological action.

Crotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus is a potent neurotoxin consisting of a weakly toxic phospholipase-A2 subunit (CB) and a non-enzymic, non-toxic subunit (CA). Crotoxin complex (CACB) dissociates upon interaction with membranes: CB binds while CA does not. Moreover, CA enhances the toxicity of CB by preventing its non-specific adsorption. Several crotoxin isoforms have been identified. Multiple variants of each subunit give different crotoxin complexes that can be subdivided into two classes: those of high toxicity and low enzymic activity and those of moderate toxicity and a high phospholipase-A2 activity. In this study, we demonstrate that the more-toxic isoforms block neuromuscular transmission of chick biventer cervicis preparations more efficiently than weakly toxic isoforms. The less-toxic crotoxin complexes have the same Km and Vmax as CB alone. In contrast, the more-toxic isoforms are enzymically less active than CB. These differences correlate with the stability of the complexes: less-toxic isoforms are less stable (Kd = 25 nM) and dissociate rapidly (half-life about 1 min), whereas the more-toxic isoforms are more stable (Kd = 4.5 nM) and dissociate more slowly (half-life 10-20 min). The rate of interaction of crotoxin complexes with vesicles of negatively charged phospholipids paralleled the rate of dissociation of the complexes in the absence of vesicles. The differences of pharmacological and biochemical properties of crotoxin isoforms indicate that the stability of crotoxin complexes plays a major role in the synergistic action of crotoxin subunits: a stronger association between the two crotoxin subunits would account for their slower dissociation rate, a weaker enzymic activity, a slower interaction with phosphatidylglycerol vesicles, a faster blockade of neuromuscular transmission and a higher lethal potency.

Animals↗
PubMed1996

Expression of an active Na+/Ca2+ exchanger isoform lacking the six C-terminal transmembrane segments.

The short isoform of the Na+/Ca2+ exchanger (67 kDa) that is produced by alternative splicing during the expression of the 6 kb canine exchanger cDNA in 293 cells was separately expressed in the same system. The protein consisted of the five N-terminal transmembrane segments and of a large portion of the main hydrophilic loop, but lacked the six C-terminal hydrophobic segments of the regular protein (108 kDa). Very high RNA levels were found after transient cell transfection with plasmid DNA encoding this truncated isoform. The RNA processing, the translation and targeting of the resulting protein to the plasma membrane appeared to be less efficient than those of the 108-kDa polypeptide produced in the same system. The Na(+)-dependent Ca(2+)-uptake activity of 293 cells expressing the short isoform was measured by an isotopic rapid filtration method, whereas the current associated with Ca2+ extrusion was measured in electrophysiological patch-clamp experiments. The results showed that the expressed isoform functioned in the typical reverse and forward Na+/Ca2+ exchange modes. In both the electrophysiological and the isotopic measurements the activity of the short isoform was 6-7-fold lower than that of the 108-kDa protein expressed in the same system. However, lower amounts of the short isoform reached the plasma membrane: its specific activity could thus be significantly higher. Possibly, the short isoform could form a dimer in which a second 67 kDa polypeptide replaces the C-terminal part of the 108-kDa protein.

Alternative Splicing↗
PubMed1997

Investigation of different recombinant isoforms of grass group-V allergens (timothy grass pollen) isolated by low-stringency cDNA hybridization--antibody binding capacity and allergenic activity.

A cDNA library of timothy grass pollen was screened for homologous isoforms of major group-V allergens by low stringency hybridization with a Phl p 5 (Phleum pratense) probe. After restriction analysis of the 40 clones obtained, 17 were selected for cDNA sequencing. Of these clones, two were unrelated to group-V allergens, six showed high similarity but an incomplete open reading frame and nine had high similarity with a complete open reading frame. Comparison of deduced amino acids of ten complete cDNA clones confirmed the presence of two major isoforms, a and b. Within these two subgroups, only minor sequence variations were observed. Eight isoforms were expressed in Escherichia coli K12 and purified to homogeneity. Although the subgroups a and b could be distinguished by their molecular masses and by binding constants towards monoclonal antibodies, all isoforms turned out to be biochemically similar. Ribonuclease activity as a marker for the biological function of group-V allergens was shown to be in the same range for both subgroups. Analysis of allergenic B-cell responses towards the isoforms in 26 grass pollen allergic patients revealed that the IgE reactivities to the different isoforms were identical for each individual. IgE reactivities and allergenic activities of three isovariants and an allergen of a different group were compared in a selected group of four grass pollen allergic patients by immunoblot, histamine-release and skin-prick tests. The IgE reactivity does not necessarily mirror the allergenic activity of the single molecule, and the variability of allergenic activity between the isovariants does not, in every case, depend on the structural differences of these allergens. We conclude that group-V isoallergens in grass pollen, although they can be structurally different, induce a similar B-cell response but can show variable allergenic activity. Thus, the most allergenic isoform of each important group of allergens should be sufficient for the diagnosis of type-I allergy. Whether the isoallergenic variation has any significant influence on the outcome of immunotherapy in allergic disease still has to be elucidated.

Allergens↗