Biomedical subjects
Publications and source records attributed to I Boime.
Intermediate lobes isolated from rat pituitary glands incorporated [35S]sulfate into pro-opiomelanocortin and other adrenocorticotropic hormone-containing peptides. Incubation of intermediate lobes in medium containing the arginine analog canavanine inhibited the cleavage of pro-opiomelanocortin into smaller products. Pro-opiomelanocortin that accumulated in the presence of canavanine was also sulfated.
Previously we showed that smooth microsomes from a variety of tissues effectively cleaved, sequestered, and "core" glycosylated nascent chains of secretory proteins. To further characterize the role of smooth membranes in the biosynthesis of secretory polypeptides, rat liver smooth microsomes were separated into smooth endoplasmic reticulum and Golgi fractions. Membranes of the smooth endoplasmic reticulum cleaved the signal peptide of pre-placental lactogen, attached the high mannose core to the alpha subunit of chorionic gonadotropin, and sequestered the processed proteins. None of these processing steps were performed by Golgi membranes. However, processing of asparagine-linked oligosaccharides and the coincident addition of terminal sugars was performed by Golgi but not by smooth endoplasmic reticulum membranes. The properties of this post-translational reaction are very similar to those described for the reactions in vivo. These observations demonstrate that the enzymes for co-translational (pre-protein processing) and posttranslational (oligosaccharide maturation) processing events are localized in the endoplasmic reticulum and Golgi apparatus, respectively. This functional differentiation of Golgi and endoplasmic reticulum membranes is an important feature of the secretory process in eukaryotic cells. Restriction of the recognition and transport of nascent secretory proteins to the endoplasmic reticulum establishes the polarity necessary for the ordered sequence of post-translational steps involved in the synthesis and maturation of secretory proteins.
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The combination of lutropin (LH; luteinizing hormone) alpha and beta subunits was examined in rat pituitaries incubated with [35S]methionine or [35S]sulfate. Combination was assessed by using antiserum directed against the beta subunit. The data show that combination of most of the subunits proceeds rapidly, well before the addition of sulfate and prior to the processing of asparagine-linked oligosaccharides to the complex form. Thus, combination appears to initiate in the endoplasmic reticulum and does not require those post-translational modifications. We observed that two forms of the LH-alpha subunit were processed--one that is secreted into the medium not associated with the LH beta subunit and another secreted as part of the alpha-beta dimer. Both forms of the alpha subunit are sulfated, and the data suggest that subsequent to sulfate addition, secretion of free alpha subunit and the dimer occur independently by separate pathways.
Probes derived from clones bearing cDNAs corresponding to the alpha subunit of human chorionic gonadotropin (hCG) and human placental lactogen (hPL) were used to localize their respective mRNAs cytologically in sections of first trimester and term human placenta. hPL mRNA was exclusively localized to the syncytial layer, hCG alpha mRNA was found in the syncytial layer and also in some differentiating cytotrophoblasts. Hybridization was specific because no signal was observed when labeled pBR322 was hybridized to placental sections or when the placental probes were hybridized to sections of human tonsils. In addition, RNA in placental interstitial cells did not hybridize with hCG alpha and hPL probes. Hybridization with the hCG alpha probe was much greater in first trimester than in term sections, whereas hPL signals were comparable in both first trimester and term placentae. Syncytial formation proceeds through cellular intermediates of cytotrophoblastic origin, and the data suggest that transcription of the hCG alpha gene is initiated before the completion of syncytial formation. In contrast, hPL mRNA synthesis starts later in trophoblast differentiation, likely after the stage of syncytial formation. The data also suggested that hCG alpha mRNA synthesis becomes attenuated but that hPL is transcribed at a rather constant rate during placental development.
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The structure of gene sequences coding for the mRNA of human chorionic gonadotropin (hCG) alpha-subunit was investigated by Southern blot analysis of genomic DNAs using a cloned, full length cDNA probe. While four hormones, lutropin, follitropin, thyrotropin, and choriogonadotropin, have homologous alpha-subunits, only one gene that bears hCG-alpha sequences could be detected per haploid complement. The structure of this single gonadotropin alpha-subunit gene, which contains intervening sequences, is the same in DNA from first trimester and term placentae. However polymorphism was observed for the presence of a HindIII site and of an Eco RI site in the gene's 3' flanking sequences. The organization of hCG-alpha sequences in several trophoblastic and nontrophoblastic tumor cell lines, which produce hCG subunits, was also examined. In each, the same gene copy number and structure were seen as in normal tissue. Thus, the characteristics of ectopic alpha-subunit expression in these cells seem not to be determined by DNA rearrangements.
Preprolactin (Pre-Prl) accumulates isolated rat pituitaries incubated with the threonine analog, beta-hydroxynorvaline. The present study examined whether the failure to cleave Pre-Prl to prolactin inhibits the translocation of the peptide chain across the membrane of the endoplasmic reticulum. In homogenates of pituitary tissue Pre-Prl, prolactin, and growth hormone were not degraded by added proteases. If 0.1% Triton X-100 was added with the proteases these proteins were degraded, suggesting that protection of these proteins from proteases was conferred by membrane vesicles. When Pre-Prl synthesized in a cell-free system was mixed and homogenized with pituitary tissue, it was completely degraded by these enzymes. Thus, protease-resistant Pre-Prl was not the result of nonspecific vesicular entrapment of free Pre-Prl during homogenization. Pre-Prl synthesized in isolated pituitaries copurified with the membrane fraction when the vesicles were isolated from tissue homogenates by flotation in 60% sucrose, and sonication and low concentrations of detergent released Pre-Prl from this fraction. Thus, removal of the prepeptide is not an essential step for the insertion of secretory proteins into the endoplasmic reticulum.
The amino acid sequences of the NH2-terminal extension prepeptides of the alpha and of the beta subunits of human choriogonadotropin have been determined by translation of first trimester placental mRNA in the presence of individually labeled amino acids. The structure of the prepeptide of the alpha subunit is: -24 -23 -22 -21 -20 -19 -18 -17 -16 -15 Met- Asp- Tyr- Tyr- Arg- Lys- Tyr- Ala- Ala- Ile -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 Phe- Leu- Val- Thr- Leu- Ser- Val- Phe- Leu- His -4 -3 -2 -1 Val- Leu- His- Ser The structure of the prepeptide of the beta subunit is: -20 -19 -18 -17 -16 -15 -14 -13 -12 -11 Met- Glu- Met- Phe- Gln- Gly- Leu- Leu- Leu- Leu -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 Leu- Leu- Leu- Ser- Met- Gly- Gly- Thr- XXX- Ala In the presence of membranes, the beta prepeptide is cleaved to a protein containing the authentic NH2-terminal sequence of the secreted form of the beta subunit. Cleavage of pre-alpha subunit also yields a product containing the NH2-terminal region of the secreted form.
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We have shown previously that processing of preprolactin to prolactin in isolated rat pituitaries is inhibited by the threonine analog, beta-hydroxynorvaline (Hnv), presumably because of its substitution for the threonine at the cleavage site. Here, we show by amino-terminal sequence analyses that Hnv altered the site at which preprolactin produced had three extra amino acids at the amino terminus. The miscleaved prolactin was secreted into the medium, and there was a delay of approximately 5 min between the initial appearance of prolactin and the formation of miscleaved prolactin. This lag period suggests that miscleaved prolactin did not represent an intermediate in the normal processing of preprolactin but resulted from cleavage of completed preprolactin chains containing Hnv. These results show that modification of the structure of a preprotein can alter the site of its cleavage. One site that appears to be critical for correct cleavage is the final amino acid residue in the prepeptide. The data also indicate that complete removal of the presequence is not required for secretion of a protein.
Chemical analyses have previously detected sulfate linked to the oligosaccharides of lutropin isolated from bovine and human pituitaries. To determine whether lutropin could be metabolically labeled with sulfate, isolated bovine and rat pituitaries were incubated with [35S]sulfate. In both species, two major labeled products were immunoprecipitated with antisera specific to lutropin subunits. Incorporation into the subunits occurred posttranslationally since it was not blocked by cycloheximide, which did, however, block the incorporation of radiolabeled methionine. Metabolic labeling with [35S]sulfate provides a valuable approach for examining the biosynthetic processing of lutropin and the physiological role of sulfate in this hormone.
The importance of threonine in the Asn-X-Thr recognition sequence for asparagine-linked glycosylation was tested by examining the effect of a threonine analog, beta-hydroxynorvaline, on co-translational glycosylation in Krebs' II ascites tumor lysates. beta-Hydroxynorvaline inhibited the glycosylation of the alpha subunit of human chorionic gonadotropin and the beta subunit of bovine luteinizing hormone; both proteins contain Asn-X-Thr recognition sites. The effect was prevented by threonine, indicating that beta-hydroxynorvaline acted via its incorporation into protein. These results provide direct evidence for the role of threonine in Asn-X-Thr sites for asparagine-linked glycosylation. The results support the view that this site is sensitive to steric hindrance.
The proteolytic processing of rat pre-prolactin (Pre-Prl) to prolactin in isolated hemipituitaries was inhibited by the threonine analog, beta-hydroxynorvaline (Hnv). Pre-Prl accumulated as a major labeled intracellular product, even during pulse labeling longer than 2 h, but it was not secreted. Inhibition of Pre-Prl processing may result from incorporation of Hnv at the Pre-Prl cleavage site. This system offers the opportunity to study the disposition of an accumulated pre-protein in cells.
The Xenopus laevis histone genes have been isolated from a bacteriophage lambda library using a cloned cDNA probe to X. laevis H4 mRNA (pX1ch4). Their structural organization has been determined by restriction mapping, blot hybridization and hybridization selection and translation of Xenopus histone mRNAs. They are clustered and probably tandemly arranged but, in contrast to invertebrate histone genes (Kedes, 1979), there has been extensive sequence divergence in the spacer regions of the clusters. Also, the order of the genes within individual clusters is not conserved. We report the isolation of two variant histone H1 gene and find that the order of the nucleosomal core histone genes within a cluster containing an H1B gene is different from that found in two clusters containing the H1A gene.
Leucine analogs were tested in the Krebs II ascites cell-free translation system for the ability to inhibit preprotein cleavage by replacing leucine in nascent chains of bovine preprolactin, rat preprolactin, human placental prelactogen (pre-hPL), and pre-alpha subunit of human chorionic gonadotropin (alpha-hCG). In the absence of analog, ascites microsomal membranes cleaved these preproteins to their mature forms and sequestered the processed products. Also, two asparagine residues in alpha-hCG were glycosylated. When 4 mM beta-DL-hydroxyleucine was added to the lysate instead of L-leucine, cotranslational processing and sequestration of both species of preprolactin and pre-hPL were inhibited. Sequential Edman degradation confirmed that pre-hPL was not cleaved. The inhibition of processing by beta-hydroxyleucine resulted from its incorporation into protein. This was shown by reversal of the effect by addition of leucine and by inhibition of [(3)H]leucine incorporation into protein. Of significance, the processing of pre-alpha-hCG was less sensitive to beta-hydroxyleucine because its prepeptide contains only four scattered leucine residues, whereas the presegments of hPL and the prolactins contain six to eight clustered leucine residues. These experiments demonstrate that translocation and processing of secretory proteins require structural features determined by the primary amino acid sequence.