Low Molecular Weight Acidic Proteins Research Articles

Purification and molecular cloning of chemosensory proteins from Bombyx mori.

Soluble low molecular weight acidic proteins are suspected to transport stimulus molecules to the sensory neurons within insect sensilla. From the antennae of Bombyx mori, we have purified and sequenced a protein (BmorCSP1) bearing sequence similarity to a class of soluble chemosensory proteins recently discovered in several orders of insects. Based on its N-terminal sequence, the cDNA encoding this protein has been amplified and cloned. Differential screening of a B. mori antennal cDNA library led to the identification of a second gene encoding a related protein (BmorCSP2), sharing 35-40% identity to BmorCSP1 and chemosensory proteins from other species. The predicted secondary structures of moth's, chemosensory proteins comprise alpha-helical foldings at conserved positions and a reduced hydrophobicity with respect to this novel family of chemosensory proteins.

Co-expression of transforming growth factor beta and interferon tau during peri-implantation period in the ewe.

The transforming growth factor beta (TGFbeta) family is known to control cell migration, growth, differentiation, function and regulation of extracellular matrix, all of which are required for the process of implantation. Expression of TGFbeta by the conceptus and endometrium was studied during the period of implantation in the ewe. A total of thirty-four ewes were hysterectomized on day 12, 14, 16, 18 or 20 of pregnancy (day 0 = day of estrus). Conceptus (200 mg wet weight) and endometrial (300 mg wet weight) tissues were cultured in vitro in 7 and 10 ml Eagle's minimal essential medium, respectively. The culture media were subjected to a bioassay to determine concentrations of TGFbeta. Conceptus culture media (CCM) were also analyzed for contents of ovine interferon-tau (oIFNr), low molecular weight acidic protein, produced by the trophectoderm between days 8 and 21 of pregnancy. Whole uteri including conceptus(es) and conceptuses (day 16) only were fixed and subjected to immunohistochemical and in situ hybridization studies. Levels of oIFNr produced by conceptuses were the highest on day 16 at 4.4 microg/ml. Concentrations of TGFbeta in day 12, 14, 16, 18 and 20 CCM were 38+/-19, 102+/-56, 862+/-152, 728+/-191 and 336+/-106 pg/ml, respectively, and approximately 90% of TGFbeta activity in CCM was due to TGFbeta1 whereas less than 10% was due to TGFbeta3 based on neutralization with TGFbeta subtype-specific antibodies. Immunohistochemical studies revealed that day 16 conceptuses displayed major staining for TGFbeta1, no beta2 staining and minor staining for beta3. In situ hybridization studies also revealed that day 16 trophectoderm possessed most TGFbeta1 mRNA while day 14 trophectoderm and day 20 chorion/amnion displayed weaker staining for TGFbeta1 mRNA. TGFbeta in day 12, 14, 16, 18 and day 20 endometrial culture media was 156+/-37, 129+/-33, 49+/-22, 62+/-23 and 179+/-40 pg/ml, respectively, and approximately 65% and 35% of the activities were due to TGFbeta1 and beta2, respectively. These results indicate that TGFbeta production by the conceptus coincides with the time when oIFNtau production starts to decline. These observations support the postulate that TGFbeta may play an important role in implantation in the ovine species.

Calcium-binding proteins in organ of Corti and basilar papilla: CBP-15, an unidentified calcium-binding protein of the inner ear

In a previous paper (Thalmann et al., 1993) we reported that the amino acid sequence of OCP2, a low molecular weight acidic protein present in extremely high concentrations in the organ of Corti and absent in the basilar papilla, exhibits a rudimentary EF-hand — a potential calcium-binding domain. The present study was undertaken to determine whether OCP2 binds 45-calcium under non-denaturing conditions following separation by isoelectric focusing and transblotting. The same criterion was used to determine whether the EF-hands of several other calcium-binding proteins (CBP) are functional in organ of Corti and basilar papilla. OCP2 exhibited no 45-calcium binding. Calmodulin, present in the organ of Corti in extremely high concentrations and lower in basilar papilla, showed strong 45-calcium binding in both structures. While calbindin represents a major protein in basilar papilla and binds 45-calcium, this protein is a minor component in the organ of Corti; whether it binds 45-calcium remains to be decided. By extending the pI range in the acidic region of isoelectric focusing, a 15 kDa, highly acidic (pI ≈ 3.1) protein was revealed that constitutes a major protein in the organ of Corti; the protein was not detectable in the basilar papilla, spiral ligament/stria vascularis complex and numerous other organs tested. It remains to be resolved whether this protein represents an isoform of parvalbumin or a novel CBP. The differential make-up of CBPs between the mammalian organ of Corti and the avian basilar papillar is discussed.

Further analysis of the cuticular proteins of the oak silkmoth and other insects

The protein extracts of the silkmoth Antherea polyphemus cuticles from larval, pupal and adult stages have been analyzed by one and two dimensional electrophoresis. While the larval cuticle consists of mainly low molecular weight acidic proteins, the majority of the pupal cuticular proteins (both wing and abdominal) exhibit sizes in the range of 15–30 kDa and isoelectric points in the range of 6.5–7.5. The adult cuticle is made up of proteins with a wider range of sizes and isoelectric points. The anti-pupal and anti-adult cuticular antibodies cross react with proteins of either stage quite well. The same antibodies do not cross react with the major larval cuticular proteins. The cuticular proteins of another silkmoth Antheraea pernyi and the well-studied tobacco hornworm Manduca sexta have also been analyzed. The electrophoretic gel patterns of the Manduca cuticular proteins and their cross reactivity with the anti-silkmoth cuticular antibodies are shown to be very similar to those seen with the A. polyphemus cuticular proteins. It is possible to distinguish the abdominal cuticular proteins as coming from a particular stage from each of the insects. The gel patterns of the cuticular proteins of the three insects from any one stage of development are much more similar to each other than those of the same insect from different stages. The protein composition of the segmental and intersegmental regions of the pupal abdomen appears similar by electrophoretic analysis.

Partial amino acid sequences of organ of Corti proteins OCP1 and OCP2: A progress report

Progress in amino acid sequencing of two low-molecular weight acidic proteins of unknown function which are present at high concentrations in the organ of Corti is reported. These two proteins, provisionally termed OCP1 and OCP2, were originally demonstrated by two-dimendional polyacrylamide gel electrophoresis; their presence at high concentrations in the inner ear sensory epithelia strongly suggests that OCP1 and OCP2 serve some important function in the ear. In the present paper, extension of the amino-terminal sequence of OCP2 to 71 residues is described. In addition, the first results on sequencing of OCP1 are presented. Computer algorithms are used to predict important structural features, and the sequences are analyzed for probable phosphorylation, glycosylation, and Ca-binding sites. Projected further studies in immunochemistry and molecular biology of OCP1 and OCP2 based on the amino acid sequencing data are discussed.

Maternal recognition of pregnancy in the goat: effects of conceptus removal on interestrus intervals and characterization of conceptus protein production during early pregnancy.

Goat conceptuses were surgically removed from the uterus at different days during early pregnancy and cultured for 24-30 h in the presence of L-[3H]leucine to determine the effects of embryo removal on the interestrus interval and to characterize in vitro synthesis and release of conceptus proteins. Normal cyclic and animals (controls) exhibited interestrus intervals of 20.44 +/- 0.89 days. Removal of conceptuses on Days 13 and 15 did not alter interestrus intervals compared to cyclic animals. Removal of conceptuses on Day 17 and times thereafter resulted in significant (p less than 0.05) prolongation of interestrus intervals. These results demonstrate that maternal recognition of pregnancy in the goat occurs between Days 15 and 17. Proteins synthesized and released into the medium by conceptuses were first detectable at Day 16 by the analytical method employed (two-dimensional polyacrylamide gel electrophoresis followed by fluorography). The major protein synthesized at this time was acidic (pI = 5.2-5.7) and consisted of two isotypes with molecular weights of about 17,000. Although patterns of protein production became more complex with conceptus development, this protein remained as a major product through Day 21 but not afterwards. This protein, as well as two other low molecular weight acidic proteins (Mr approximately equal to 21,000, 23,000; pI = 5.7-6.0) were shown by immunoprecipitation to react with anti-ovine trophoblast protein-1 (oTP-1) serum. Hence, these products may comprise a caprine trophoblast protein-1 (cTP-1) complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of bovine conceptus proteins produced during the peri- and postattachment periods of early pregnancy.

Bovine conceptuses removed from the uterus during the peri- and postattachment periods of placentation (Days 17-24 and 26-38, respectively) were cultured in a modified minimum essential medium in the presence of L-[3H]leucine to characterize in vitro synthesis of proteins released into the medium. Patterns of protein production were analyzed by two-dimensional polyacrylamide gel electrophoresis followed by fluorography of dried gels. Four groups of low molecular weight acidic proteins (LMWAP) were observed to be synthesized during the peri- and postattachment periods. The number and relative concentrations of these changed with development. One group (A) consisted of three major and two or more minor isoelectric species (pI approximately equal to 5.8-6.8); these were the major synthesized proteins observed from Days 17-22. The major polypeptides of Group A were present at all time points examined through Day 38 and, in several preparations, appeared as doublets (Mr approximately equal to 22,000 and 24,000) through Day 29 but not thereafter. Group A polypeptides from Day 19 and 36 conceptus cultures were demonstrated by immunoblot analysis to cross-react with antiserum produced against ovine trophoblast protein-1 (oTP-1). A second group of proteins (A1) and a single protein (B) in the 20,000-24,000 Mr range were observed between Days 17 and 22. These were acidic relative to Group A and were not detected after Day 22. A fourth group (C) of LMWAP (Mr approximately equal to 14,000-18,000) was first observed around Day 21 and appeared to increase relative to Group A through Day 29. One protein from this group, C3, was the predominant LMWAP at Day 38.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel proteins belonging to the troponin C superfamily are encoded by a set of mRNAs in sea urchin embryos

The properties of several cDNA clones representing a family of mRNAs found in the embryonic ectoderm of Strongylocentrotus purpuratus are described. We have previously shown that these mRNAs (termed Spec for Strongylocentrotus purpuratus ectoderm) accumulate in the presumptive dorsal ectoderm of post-cleavage stage embryos and code for a group of 10 to 12 low molecular weight acidic proteins. We demonstrate here, using antibodies raised against the major Spec proteins, that the proteins are localized in the cytoplasm of dorsal ectoderm cells. Hybridization analysis and DNA sequencing show that the mRNAs coding for these proteins, although all related, can be divided into two subfamilies. Comparison of the translational reading frames of the Spec mRNAs with known protein sequences shows a significant homology with troponin C-related proteins, especially in the calcium-binding domains. We suggest that the Spec proteins are previously uncharacterized members of the troponin C superfamily.

Increased synthesis of a low molecular weight protein in vincristine-resistant cells

A 19,000-dalton peptide (pI = 5.7) that is synthesized in increased amounts in vincristine-resistant Chinese hamster cells ( DC-3F VCRd-5 ) has been identified by two-dimensional gel electrophoresis. Reduced amounts of the protein were present in a revertant line of DC-3F VCRd-5 , and only trace amounts were detected in control DC-3F cells. A similar protein (M r = 19,000; pI = 5.7) was also found in a vincristine-resistant mouse line. Two vincristine-resistant human neuroblastoma cell lines likewise contained elevated levels of a low molecular weight acidic protein. Increased biosynthesis of the 19,000-dalton polypeptide in DC-3F VCRd-5 cells coincides with the presence of a homogeneously staining region, HSR, on a metaphase chromosome.

Support for chromatin acidic proteins as acceptors for progesterone in the chick oviduct

Studies in this laboratory have supported the role of chromosomal protein-DNA complexes as the nuclear acceptor sites for progesterone in the avian oviduct. The protein coacceptor appears to be a low molecular weight acidic protein(s) which when removed from the DNA results in a marked loss of binding by the activated progesterone-receptor complex. When the protein is reannealed back to the pure DNA, the binding capacity is restored. During studies on these nuclear binding sites for progesterone in the hen oviduct, a seasonal variation in the level and function of the progesterone receptor (P-R) was detected. Cytosol preparations obtained from the chick oviducts during the winter/ spring period between January and May display reduced receptor levels as well as a loss of the capacity of the receptor to bind to nuclear “acceptor” sites in vitro. The binding of [ 3H]-P-R to whole chromatin or purified acceptor proteins reannealed to DNA display the same rhythm. No such rhythm is detected for the binding of P-R to pure DNA. The nuclear binding in vivo, achieved by injecting [ 3H]-progester-one into the wing vein and analyzing the radioactivity localized in the oviduct nuclei, also displays a similar rhythm. These results indicate that the native nuclear acceptor sites for progesterone in the chick oviduct are protein-DNA complexes and not pure DNA. The failure of P-R to bind the nuclear acceptor sites in vivo and in vitro during this period can be explained by the two subunit hypothesis of Schrader and O'Malley, whereby one of the two subunits is absent or inactive during this period.

Proteins of cholinergic synaptic vesicles from the electric organ of Torpedo: Characterization of a low molecular weight acidic protein

A study has been made, using gel filtration, of the membrane-bound and soluble proteins obtained when cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata and related species are exposed to water. The usual technique for releasing protein was to dialyze suspensions of vesicles separated by zonal centrifuging in distilled water; the freeze-dried residue was then filtered through columns of Biol-Gel A-5 m or Sephadex G-200. About 60% of the protein was recovered in a void volume fraction. On disc-gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), this fraction gave 3 prominent high molecular weight components. Over 40% of the protein was a low molecular weight, acidic protein which we have called vesiculin. Hydrolysates were rich in hydroxy amino acids as well as glutamate and aspartate. Vesiculin is loosely associated with a small molecule, probably a nucleotide or mixture of nucleotides. Estimates of molecular weight by amino acid analysis, disc-gel electrophoresis, gel filtration and sedimentation coefficient all indicate a value close to 10,000. Vesiculin differs in composition and molecular weight from other acidic brain proteins. It is suggested that vesiculin may constitute the counter-ion to acetylcholine within the vesicle core.

Glycine reduction to acetate and ammonia: Identification of ferredoxin and another low molecular weight acidic protein as components of the reductase system

Protein A, one of the catalytic components of the glycine reductase system of Clostridium sticklandii and related amino acid-fermenting clostridia, was partially purified and characterized as an acidic low molecular weight protein apparently possessing one or more sulfhydryl groups essential for activity. Certain crude extracts unable to reduce glycine unless supplemented with protein A can be shown to require this protein irrespective of the electron donor system employed, i.e., with reduced methyl viologen, a DPNH-generating system or with dimercaptans such as 1,4-dimercaptothreitol. In contrast, ferredoxin and another unidentified factor(s) also removed by treatment of extracts with DEAE-cellulose are required for glycine reduction in the DPNH-linked system but are bypassed when dimercaptans serve as electron donors.