Structural and Biological Characterization of Two Crotamine Isoforms IV-2 and IV-3 Isolated from the <Emphasis Type="Italic">Crotalus durissus cumanensis</Emphasis> Venom

In this work, we isolated the two new crotamine isoforms from the Crotalus durissus cumanensis rattlesnake venom and its “in vitro” neurotoxic, myotoxic and lethality (DL₅₀) intracerebroventricular (i.c.v.) effects were characterized. These proteins were named IV-2 and IV-3 and were purified by combination of two chromatographic steps on molecular exclusion chromatography on Superdex 75 and reverse phase HPLC (μ-Bondapack C18). The molecular mass of the crotamine isoforms was 4905.96 Da for isoform IV-2 and 4956.97 Da for IV-3 and, as determined by mass spectrometry, and both contained six Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of both isoforms. The positions of five sequenced tryptic peptides, including the N-terminal of the isoform IV-2 and four from isoform IV-3 were deduced by comparison with a homologous protein from the crotamine family. The isoforms IV-2 and IV-3 had a sequence of amino acids of 42 amino acid residues IV-2: YKRCHIKGGH CFPKEKLICI PPSSDIGKMD CPWKRKCCKK RS and pI value 9.54 and IV-3: YKQCHKKGGH CFPKEVLICI PPSSDFGKMD CRWKRKCCKK RS with a pI value of 9.54. This protein showed high molecular amino acid sequence identity with other crotamine-like proteins from Crotalus durissus terrificus. These new crotamine isoforms induced potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and potent myotoxic effect. In mice, both isoforms induced myonecrosis, upon intramuscular or subcutaneous injections. These activities were modulated by the presence of positively charged amino acid residues. The LD₅₀ of isoform IV-2 was 0.07 mg/kg and isoform IV-3 was 0.06 mg/kg the animal weight, by i.c.v. route.     

Vacuolar-type H<Superscript>+</Superscript>-ATPase with the <Emphasis Type="Italic">a</Emphasis>3 isoform is the proton pump on premature melanosomes

The melanosome, an organelle specialized for melanin synthesis, is one of the lysosome-related organelles. Its lumen is reported to be acidified by vacuolar-type H⁺-ATPase (V-ATPase). Mammalian V-ATPase exhibits structural diversity in its subunit isoforms; with regard to membrane intrinsic subunit a, four isoforms (a1–a4) have been found to be localized to distinct subcellular compartments. In this study, we have shown that the a3 isoform is co-localized with a melanosome marker protein, Pmel17, in mouse melanocytes. Acidotropic probes (LysoSensor and DAMP) accumulate in non-pigmented Pmel17-positive melanosomes, and DAMP accumulation is sensitive to bafilomycin A1, a specific inhibitor of V-ATPase. However, none of the subunit a isoforms is associated with highly pigmented mature melanosomes, in which the acidotropic probes are also not accumulated. oc/oc mice, which have a null mutation at the a3 locus, show no obvious defects in melanogenesis. In the mutant melanocytes, the expression of the a2 isoform is modestly elevated, and a considerable fraction of this isoform is localized to premature melanosomes. These observations suggest that the V-ATPase keeps the lumen of premature melanosomes acidic, whereas melanosomal acidification is less significant in mature melanosomes. Ge-Hong Sun-Wada and Yoh Wada contributed equally to this study. This study was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by the Hayashi and Noda Foundations.     

Differential intranuclear localization of fibroblast growth factor-2 isoforms and specific interaction with the survival of motoneuron protein

Fibroblast growth factor 2 (FGF-2) is an important modulator of cell growth and differentiation and a neurotrophic factor. FGF-2 occurs in isoforms, at a low molecular weight of 18,000 and at least two high molecular weight forms (21,000 and 23,000), representing alternative translation products from a single mRNA. In addition to its role as an extracellular ligand, FGF-2 localizes to the nuclei of cells. Here we show differential localization of the 18- and 23-kDa isoforms in the nuclei of rat Schwann cells. Whereas the 18-kDa isoform was found in the nucleoli, nucleoplasm, and Cajal bodies, the 23-kDa isoform localized in a punctuate pattern and associates with mitotic chromosomes suggesting different functional roles of the isoforms. Moreover, we show here that the 23-kDa FGF-2 isoform co-immunoprecipitates specifically with the survival of motor neuron protein (SMN). SMN is an assembly and recycling factor of the splicing machinery and locates to the cytoplasm, the nucleoplasm, and nuclear gems, where it co-localizes with 23-kDa FGF-2. Patients with spinal muscular atrophy suffer from fatal degeneration of motoneurons because of mutations and deletions of the gene for the SMN protein.     

Inhibition studies with rationally designed inhibitors of the human low molecular weight protein tyrosine phosphatase

The human low molecular weight protein tyrosine phosphatase (HCPTP) is ubiquitously expressed as two isoforms in a wide range of human cells and may be involved in regulating the metastatic nature of epithelial tumors. A homology model is presented for the HCPTP-B isoform based on known X-ray crystal structures of other low molecular weight PTPs. A comparison of the two isoform structures indicates the possibility of developing isoform-specific inhibitors of HCPTP. Molecular dynamics simulations with CHARMM have been used to study the binding modes of the known adenine effector and phosphate in the active site of both isoforms. This analysis led to the design of the initial lead compound, based on an azaindole ring moiety, which was then also evaluated computationally. A comparison of these simulations indicates the need for a phosphonate group on the indole and provides insight into inhibitor binding modes. Compounds with varying degrees of structural similarity to the azaindole have been synthesized and tested for inhibition with each isoform. These molecular systems were examined with the program AutoDock, and comparisons made with the kinetics and the explicit simulations to validate AutoDock as a screening tool for potential inhibitors. Two compounds were experimentally found to have sub-millimolar inhibition, but the greater solubility of one reinforces the need for experimental testing alongside computational analysis.     

High molecular weight isoforms of growth hormone in cells of the immune system

A substantial body of research exists to support the idea that cells of the immune system produce growth hormone (GH). However, the structure and mechanism of action of lymphocyte-derived GH continues to remain largely unknown. Here we present the results of Western analysis of whole cell extracts showing that different molecular weight isoforms of GH of approximately 100, 65, and 48 kDa can be detected in primary mouse cells of the immune system and in the mouse EL4 cell line. The identity of the 65 and 48 kDa isoforms of GH were confirmed by mass spectrometry. The various isoforms were detected in both enriched T and B spleen cell populations. The large molecular weight isoform appears to reside primarily in the cytoplasm, whereas the lower molecular weight 65 and 48 kDa isoforms were detected primarily in the nucleus. These results also suggest that GH isoforms are induced by oxidative stress. In EL4 cells overexpressing GH, the expression of luciferase controlled by a promoter containing the antioxidant response element is increased almost threefold above control. The data suggest that the induction of isoforms of the GH molecule in cells of the immune system may be an important mechanism of adaptation and/or protection of lymphoid cells under conditions of oxidative stress.     

Effect of overexpressing fibroblast growth factor 2 protein isoforms in osteoblastic ROS 17/2.8 cells

Fibroblast growth factor-2 (FGF-2) is made by osteoblasts and modulates their function. There are high molecular weight (HMW) protein isoforms of FGF-2 that have nuclear localization sequences and a low molecular weight (LMW) 18 kDa FGF-2 protein that is exported from cells. Since FGF-2 is a trophic factor and potent mitogen for osteoblasts, the goal of this study was to utilize targeted overexpression of FGF-2 as a novel means of assessing different FGF-2 isoforms on osteoblastic cell viability and proliferation. Either LMW or HMW human Fgf2 cDNAs were cloned downstream of 3.6 kb alpha1(I)-collagen 5' regulatory elements (Col 3.6). A set of expression vectors, called Col3.6-Fgf2 isoforms-IRES-GFPsaph, capable of concurrently overexpressing either LMW or HMW FGF-2 isoforms concomitant with GFPsaph from a single bicistronic mRNA were built. Viable cell number in ROS 17/2.8 cells stably transfected with Vector (Col3.6-IRES-GFPsaph) versus each of the Col3.6-Fgf2-IRES-GFPsaph constructs were compared. In the presence of 1 or 10% serum, DNA synthesis was increased in cells expressing any isoform of FGF-2 compared with vector. However, cells transfected with HMW isoform had augmented DNA synthesis in 1 or 10% serum compared with cells expressing either ALL or LMW FGF-2 isoforms. A neutralizing FGF-2 antibody significantly reduced the mitogenic response in cells harboring ALL or the LMW FGF-2 isoforms but did not block the mitogenic effect of cells harboring the HMW isoforms. In summary, overexpression of any isoform of FGF-2 protein increased viable cell number and OB proliferation in the presence of low or high concentrations of serum. However, the HMW/nuclear isoforms preferentially mediate augmented OB proliferation. We conclude that differential expression of FGF-2 proteins isoforms is important in modulating OB function.     

Identification and characterization of a novel,intracellular isoform of fibroblast growth factor receptor-1 (FGFR-1)

A novel, low molecular weight, intracellular isoform of FGF receptor-1 (FGFR-1) was identified in embryonic chicken tissues using several antibodies specific for different domains of FGF receptors. This low molecular weight isoform differs from the previously characterized isoforms of FGFR-1 in that it contains little or no carbohydrate. Furthermore, in contrast to the other isoforms of FGFR-1, this novel isoform is located exclusively intracellularly. However, it is capable of binding ¹²⁵I-FGF-2 and it possesses intrinsic kinase activity. Pulse-chase experiments indicate that this isoform of FGFR-1 is not simply a precursor to glycosylated FGFR-1 since it can be detected long after the appearance of glycosylated FGFR-1 in the cells. These results suggest that the novel FGFR-1 isoform plays a role in regulating FGF activity distinct from cell surface, glycosylated FGFR-1. The possible roles of this FGFR-1 variant in FGF signaling are discussed. © 1996 Wiley-Liss, Inc.     

Isoformes of Malate Dehydrogenase from <Emphasis Type="Italic">Rhodovulum Steppense</Emphasis> A-20s Grown Chemotrophically under Aerobic Conditions

Three malate dehydrogenase isoforms (65-, 60-, and 71-fold purifications) with specific activities of 4.23, 3.88, and 4.56 U/mg protein were obtained in an electrophoretically homogenous state from Rhodоvulum steppense bacteria strain A-20s chemotrophically grown under aerobic conditions. The physicochemical and kinetic properties of malate dehydrogenase isoforms were determined. The molecular weight and the Michaelis constants were determined; the effect of hydrogen ions on the forward and reverse MDH reaction was studied. The results of the study demonstrated that the enzyme consists of subunits; the molecular weight of subunits was determined by SDS-PAGE.     

Alpha-actinin expression during avian myogenesis in vivo. Evidence for the existence of an embryo-specific isoform of alpha-actinin

The isoforms of skeletal muscle alpha-actinin present during chick embryogenesis were analyzed by two-dimensional electrophoresis in combination with the immunoblot technique. Chicken embryonic muscles at 8-15 days contain an embryo-specific isoform of alpha-actinin. The embryonic alpha-actinin isoform has a molecular mass of 112 kDa and an isoelectric point of 5.8, whereas the values for the adult isoform of alpha-actinin were 100 kDa and 5.85, respectively. To characterize the two classes of alpha-actinin polypeptides we have compared the two proteins by 125I-labeled two-dimensional peptide mapping. The embryonic isoform is highly similar to, but exhibited extensive peptide differences to, the adult isoform of alpha-actinin. The developmental sequence of the expression of the alpha-actinins was also studied. In extracts of skeletal muscle from 8-10-day-old embryos, only the embryonic isoform was detected. In extracts from 15-day-old embryos, both the embryonic and the adult isoforms coexisted. However by 21 days, the embryonic isoform had disappeared and only the adult isoform was detected. These data suggested that the embryonic and the adult isoform of alpha-actinins are distinct proteins and that during skeletal myogenesis in ovo one class of alpha-actinin is replaced by a new class of alpha-actinin polypeptides, and that the latter is maintained into adulthood.     

Isoform purification of gastric lipases. Towards crystallization.

Several isoforms of rabbit and human gastric lipases have been purified. These isoforms have the same apparent molecular weight (Mr approximately 50,000), but very different isoelectric points. Some of these isoforms were purified: pI 7.2 and 6.5 in the case of rabbit gastric lipase; and pI 7.4 and 7.2 in that of human gastric lipase. All the purified isoforms were found to have the same specific lipase activity (around 1200 units per mg of protein, measured on tributyrin as substrate). The isoforms of dog gastric lipase are more closely related, and could not be separated. Partial enzymatic deglycosylation of human gastric lipase reduced the apparent molecular weight from Mr approximately 50,000 to Mr approximately 43,000 and induced a change in the isoelectrofocusing pattern and the emergence of a new isoform (pI 7.3). It is concluded that the charge heterogeneity of gastric lipases is at least partly due to the glycan moiety of the molecule, which amounts to approximately 14% of the total molecular weight. Several crystallization trials on purified native preparations of rabbit and human gastric lipases were unsuccessful, whereas crystals were obtained from native dog gastric lipase and all the purified isoforms of rabbit and human gastric lipases, some of which were crystallographically characterized.     

Lamina-associated polypeptide 2 (LAP2) expression in fish and amphibians

Somatic and germinal cells of 15 fish and 33 amphibian species were examined by SDS-PAGE followed by immunoblotting to determine the expression of LAP2 (lamina-associated polypeptide 2). LAP2 expression in frogs, salamanders and fish does not vary with the mode of reproduction. In fish and frog cells, a rim-like LAP2 positive region was detected around the nucleus by indirect immunofluorescence microscopy. The cell distribution and expression patterns of LAP2 in fish, frogs and salamanders are comparable with those found in Xenopus and zebrafish. The mammalian somatic cell pattern, which may also occur in gymnophione amphibians, includes LAP2alpha, beta and gamma as major isoforms, whereas LAP2alpha does not occur in cells of fish, frogs and salamanders. In fish, LAP2gamma is the major isoform of somatic cells, suggesting that LAP2gamma may be ancestral. However, in the rainbow trout, as in frogs and salamanders, LAP2beta was the major somatic isoform. Fish and frog sperm only express low molecular weight polypeptides. In contrast, fish and frog oocytes express an oocyte-specific LAP2 isoform of high molecular weight. In the toad Bufo marinus this isoform becomes upregulated in pre-vitellogenic oocytes of 150-200 microm in diameter. The absence of LAP2alpha and the differential expression of LAP2 isoforms in somatic and germ cells, as found in fish and frogs, may be ancestral vertebrate characters. In spite of differences in developmental time, the LAP2 isoforms of somatic cells are upregulated during gastrulation, suggesting that LAP2 may be implicated in the early development of fish and frog.     

In Vivo Neuronal Synthesis and Axonal Transport of Kunitz Protease Inhibitor (KPI)-Containing Forms of the Amyloid Precursor Protein

Abstract: We have shown previously that the amyloid precursor protein (APP) is synthesized in retinal ganglion cells and is rapidly transported down the axons, and that different molecular weight forms of the precursor have different developmental time courses. Some APP isoforms contain a Kunitz protease inhibitor (KPI) domain, and APP that lacks the KPI domain is considered the predominant isoform in neurons. We now show that, among the various rapidly transported APPs, a 140-kDa isoform contains the KPI domain. This APP isoform is highly expressed in rapidly growing retinal axons, and it is also prominent in adult axon endings. This 140-kDa KPI-containing APP is highly sulfated compared with other axonally transported isoforms. These results show that APP with the KPI domain is a prominent isoform synthesized in neurons in vivo, and they suggest that the regulation of protease activity may be an important factor during the establishment of neuronal connections.     

Isolation of a new laccase isoform from the white-rot fungi Pycnoporus cinnabarinus strain ss3

Two extracellular laccase isoforms (Lac I and Lac II) produced by the white-rot fungus Pycnoporus cinnabarinus from the monokaryotic strain ss3 were purified from ferulic-acid-induced liquid culture medium using ammonium sulphate precipitation, followed by anion-exchange chromatography on a Mono Q column. Strain ss3 is the first generation of the parental strain P. cinnabarinus I-937. The new isolated isoform, Lac II, consists of an 86,000 molecular weight protein as determined by SDS polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of both isoforms were determined, and compared to known laccase protein sequences of other organisms.     

Investigations of Molecular Mechanisms of Actin–Myosin Interactions in Cardiac Muscle

The functional characteristics of cardiac muscle depend on the composition of protein isoforms in the cardiomyocyte contractile machinery. In the ventricular myocardium of mammals, several isoforms of contractile and regulatory proteins are expressed–two isoforms of myosin (V1 and V3) and three isoforms of tropomyosin chains (α, β, and κ). Expression of protein isoforms depends on the animal species, its age and hormonal status, and this can change with pathologies of the myocardium. Mutations in these proteins can lead to cardiomyopathies. The functional significance of the protein isoform composition has been studied mainly on intact hearts or on isolated preparations of myocardium, which could not provide a clear comprehension of the role of each particular isoform. Present-day experimental techniques such as an optical trap and in vitro motility assay make it possible to investigate the phenomena of interactions of contractile and regulatory proteins on the molecular level, thus avoiding effects associated with properties of a whole muscle or muscle tissue. These methods enable free combining of the isoforms to test the molecular mechanisms of their participation in the actin–myosin interaction. Using the optical trap and the in vitro motility assay, we have studied functional characteristics of the cardiac myosin isoforms, molecular mechanisms of the calcium-dependent regulation of actin–myosin interaction, and the role of myosin and tropomyosin isoforms in the cooperativity mechanisms in myocardium. The knowledge of molecular mechanisms underlying myocardial contractility and its regulation is necessary for comprehension of cardiac muscle functioning, its disorders in pathologies, and for development of approaches for their correction.     

Evidence of the Involvement of Asparagine Deamidation in the Formation of Cyclodextrin Glycosyltransferase Isoforms in <Emphasis Type="Italic">Paenibacillus</Emphasis> sp. RB01

Cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. RB01 and its recombinant enzyme exhibit three isoforms (I, II, and III) with the same apparent size but different charge. Here, we demonstrate for the first time that the deamidation of labile Asns causes the change in molecular forms of CGTase. The faster increase in number of isoforms was observed upon incubation in deamidation buffer at the more alkaline pH. The increase in levels of isoform II and III over time correlated with the increase in isoaspartate, a unique deamidation product. The predicted labile Asns were individually mutated to Asp, then the selected mutant and wild type isoforms were tryptic digested and labile Asns were investigated by MALDI-TOF. From the results, Asn427 was the most susceptible residue for deamidation, followed by Asn336, Asn415, and Asn567. In addition, Gln389 might also share a role. The advantage of using appropriate CGTase isoform in cyclodextrin production is reported.     

Mass spectrometry and structural characterization of 2S albumin isoforms from Brazil nuts (Bertholletia excelsa)

Proteomic approaches have been used to characterise the main 2S albumin isoforms from Brazil nuts (Bertholletia excelsa). Whilst most isoforms ( approximately 10 discrete protein species) exhibited molecular masses of around 12 kDa with a high amino acid sequence homology, important charge heterogeneity was found, with pIs varying between 4.6 and 6.6, with one >or=7.0. Proteomic analysis showed that these corresponded to a total of six National Center for Biotechnology Information (NCBI) accessions and that three isoforms had been purified to homogeneity corresponding to gi/384327, 112754 and 99609. The latter sequence corresponds to an isoform, previously only identified at the nucleotide sequence level, had a slightly higher molecular weight (13.4 kDa), and with noticeable differences in the primary structure. Proteins corresponding to six different NCBI accessions were identified, the heterogeneity of which had been increased by posttranslational processing. Evidence was found of cyclization of the N-terminal glutamine residue in two isoforms, together with ragged C-termini, indicative of carboxypeptidase activity within the vacuole following posttranslational processing. No evidence of glycosylation was found. Circular dichroism (CD) and Fourier transform-infrared (FT-IR) spectroscopy indicated all the studied isoforms were predominantly alpha-helical in nature, but that the Mr 13400 species was structurally distinct, with a higher proportion of alpha-helical structure.