scholarly journals Translational control of an intestinal microvillar enzyme

1986 ◽  
Vol 235 (2) ◽  
pp. 447-451 ◽  
Author(s):  
E M Danielsen ◽  
G M Cowell ◽  
H Sjöström ◽  
O Norén
Keyword(s):  
Translational Control ◽  
Aminopeptidase N ◽  
Total Rna ◽  
Membrane Bound ◽  
High Mannose ◽  
Small Intestinal ◽  
Adult Rate ◽  
Foetal Intestine ◽  
Translational Level

The rates of biosynthesis of adult and foetal pig small-intestinal aminopeptidase N (EC 3.4.11.2) were compared to determine at which level the expression of the microvillar enzyme is developmentally controlled. In organ-cultured explants, the rate of biosynthesis of foetal aminopeptidase N is only about 3% of the adult rate. The small amount synthesized occurs in a high-mannose-glycosylated, membrane-bound, form that is processed to the mature, complex-glycosylated, form at a markedly slower rate than that of the adult enzyme. Extracts of total RNA from adult and foetal intestine contained comparable amounts of aminopeptidase N mRNA, encoding gel-electrophoretically identical primary translation products. Together, these data indicate that the expression of aminopeptidase N is controlled at a translational level.

scholarly journals Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV

1983 ◽  
Vol 210 (2) ◽  
pp. 389-393 ◽  
Author(s):  
E M Danielsen ◽  
H Sjöström ◽  
O Norén
Keyword(s):  
Organ Culture ◽  
Membrane Fraction ◽  
Bound State ◽  
Dipeptidyl Peptidase ◽  
Dipeptidyl Peptidase Iv ◽  
Soluble Form ◽  
Membrane Bound ◽  
Aminopeptidase A ◽  
High Mannose ◽  
Small Intestinal

The biogenesis of three intestinal microvillar enzymes, maltase-glucoamylase (EC 3.2.1.20), aminopeptidase A (aspartate aminopeptidase, EC 3.4.11.7) and dipeptidyl peptidase IV (EC 3.4.14.5), was studied by pulse-chase labelling of pig small-intestinal explants kept in organ culture. The earliest detectable forms of the enzymes were polypeptides of Mr 225000, 140000 and 115000 respectively. These were found to represent the enzymes in a ‘high-mannose’ state of glycosylation, as judged by their susceptibility to treatment with endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96). After about 40-60 min of chase, maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV were further modified to yield the mature polypeptides of Mr 245000, 170000 and 137000 respectively, which were expressed at the microvillar membrane after 60-90 min of chase. The fact that the enzymes before reaching the microvillar membrane were found in a Ca2+-precipitated membrane fraction (intracellular and basolateral membranes), but not in soluble form, indicates that during biogenesis maltase-glucoamylase, aminopeptidase A and dipeptidyl peptidase IV are transported and assembled in a membrane-bound state.

scholarly journals Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on aminopeptidase N and sucrase-isomaltase

1982 ◽  
Vol 204 (3) ◽  
pp. 639-645 ◽  
Author(s):  
E M Danielsen
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Plasma Membrane ◽  
Aminopeptidase N ◽  
Secretory Proteins ◽  
Oligosaccharide Structure ◽  
Membrane Flow ◽  
Polypeptide Synthesis ◽  
High Mannose ◽  
Small Intestinal

The biogenesis of two microvillar enzymes, aminopeptidase N (EC 3.4.11.2) and sucrase (EC 3.2.1.48)-isomaltase (EC 3.2.1.10), was studied by pulse-chase labelling of pig small-intestinal explants kept in organ culture. Both enzymes became inserted into the membrane during or immediately after polypeptide synthesis, indicating that translation takes place on ribosomes attached to the rough endoplasmic reticulum. The earliest detectable forms of aminopeptidase and sucrase-isomaltase were polypeptides of Mr 140 000 and 240 000 respectively. These polypeptides were susceptible to treatment with endo-β-N-acetylglucosaminidiase H (EC 3.2.1.96), suggesting that the microvillar enzymes during or immediately after completion of protein synthesis become glycosylated with a ‘high-mannose’ oligosaccharide structure similarly to other plasma-membrane and secretory proteins. After 20-40 min or 60-90 min of chase, respectively, aminopeptidase N and sucrase-isomaltase were reglycosylated to give the polypeptides of Mr 166 000 (aminopeptidase N) and 265 000 (sucrase-isomaltase). These were expressed at the microvillar membrane after 60-90 min. During the entire process of synthesis and transport to the microvillar membrane the enzymes were bound to membranes, indicating that the biogenesis of aminopeptidase N and sucrase-isomaltase occurs in accordance with the membrane flow hypothesis.

scholarly journals Biosynthesis of intestinal microvillar proteins. The effect of swainsonine on post-translational processing of aminopeptidase N

1983 ◽  
Vol 216 (2) ◽  
pp. 325-331 ◽  
Author(s):  
E M Danielsen ◽  
G M Cowell ◽  
O Norén ◽  
H Sjöström ◽  
P R Dorling
Keyword(s):  
Palmitic Acid ◽  
Golgi Complex ◽  
Alkaline Hydrolysis ◽  
Considerable Increase ◽  
Aminopeptidase N ◽  
Final Destination ◽  
High Mannose ◽  
Small Intestinal ◽  
Mucosal Explants

The post-translational processing of pig small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in organ-cultured mucosal explants. Exposure of the explants to swainsonine, an inhibitor of Golgi mannosidase II, resulted in the formation of a Mr-160000 polypeptide, still sensitive to endo-beta-N-acetylglucosaminidase H. Swainsonine caused only a moderate inhibition of transport of the enzyme through the Golgi complex and the subsequent expression in the microvillar membrane. This may imply that the trimming of the high-mannose core and complex glycosylation of N-linked oligosaccharides is not essential for the transport of aminopeptidase N to its final destination. A different type of processing was observed to take place in the presence of swainsonine, resulting in a considerable increase in apparent Mr (from 140000 to 160000). This processing could not be ascribed to N-linked glycosylation, since treatment of the Mr-160000 polypeptide with endo-beta-N-acetylglucosaminidase H only decreased its apparent Mr by 15000. The susceptibility of the mature Mr-166000 polypeptide, but not the Mr-140000 polypeptide, to mild alkaline hydrolysis suggests that aminopeptidase N becomes glycosylated with O-linked oligosaccharides during its passage through the Golgi complex. Aminopeptidase N was not labelled by [3H]palmitic acid, indicating that the processing of the enzyme does not include acylation.

scholarly journals Biosynthesis of intestinal microvillar proteins. Processing of aminopeptidase N by microsomal membranes

1983 ◽  
Vol 212 (1) ◽  
pp. 161-165 ◽  
Author(s):  
E M Danielsen ◽  
O Norén ◽  
H Sjöström
Keyword(s):  
Signal Sequence ◽  
Aminopeptidase N ◽  
Soluble Form ◽  
Translation System ◽  
Translation Product ◽  
Free Translation ◽  
Microsomal Membranes ◽  
A Cell ◽  
Membrane Bound ◽  
Small Intestinal

The biosynthesis of small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in a cell-free translation system derived from rabbit reticulocytes. When dog pancreatic microsomal fractions were present during translation, most of the aminopeptidase N synthesized was found in a membrane-bound rather than a soluble form, indicating that synthesis of the enzyme takes place on ribosomes attached to the rough endoplasmic reticulum. The microsomal fractions process the Mr-115 000 polypeptide, which is the primary translation product of aminopeptidase N, to a polypeptide of Mr 140 000. This was found to be sensitive to the action of endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96), showing that aminopeptidase N undergoes transmembrane glycosylation during synthesis. The position of the signal sequence in aminopeptidase N was determined by a synchronized translation experiment. It was found that microsomal fractions should be added before about 25% of the polypeptide was synthesized to ensure processing to the high-mannose glycosylated form. This suggests that the signal sequence is situated in the N-terminal part of the aminopeptidase N. The size of the cell-free translation product in the absence of microsomal fractions was found to be similar to that on one of the forms of the enzyme obtained from tunicamycin-treated organ-cultured intestinal explants.

scholarly journals Posttranscriptional control of embryonic rat skeletal muscle protein synthesis. Control at the level of translation by endogenous RNA.

1988 ◽  
Vol 107 (3) ◽  
pp. 1085-1098 ◽  
Author(s):  
C R Vanderburg ◽  
M A Nathanson
Keyword(s):  
Skeletal Muscle ◽  
Cell Differentiation ◽  
Muscle Cell ◽  
Translational Control ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Skeletal Muscle Protein ◽  
Rat Skeletal Muscle ◽  
Total Rna ◽  
Muscle Cell Differentiation

The onset of muscle cell differentiation is associated with increased transcription of muscle-specific mRNA. Studies from this laboratory using 19-d embryonic rat skeletal muscle, suggest that additional, posttranscriptional controls regulate maturation of muscle tissue via a quantitative effect upon translation, and that the regulatory component may reside within the poly A- RNA pool (Nathanson, M.A., E.W. Bush, and C. Vanderburg. 1986. J. Biol. Chem. 261:1477-1486). To further characterize muscle cell translational control, embryonic and adult total RNA were separated into oligo(dT)cellulose-bound (poly A+) and -unbound (poly A-) pools. Unbound material was subjected to agarose gel electrophoresis to resolve constituents of varying molecular size and mechanically cut into five fractions. Material of each fraction was electroeluted and recovered by precipitation. Equivalent loads of total RNA from 19-20-d embryonic rat skeletal muscle exhibited a 40% translational inhibition in comparison to its adult counterpart. Inhibition was not due to decreased message abundance because embryonic, as well as adult muscle, contained equivalent proportions of poly A+ mRNA. An inhibition assay, based upon the translatability of adult RNA and its inhibition by embryonic poly A- RNA, confirmed that inhibition was associated with a 160-2,000-nt poly A- fraction. Studies on the chemical composition of this fraction confirmed its RNA composition, the absence of ribonucleoprotein, and that its activity was absent from similarly fractionated adult RNA. Rescue of inhibition could be accomplished by addition of extra lysate or mRNA; however, smaller proportions of lysate were required, suggesting a strong interaction of inhibitor and components of the translational apparatus. Additional studies demonstrated that the inhibitor acted at the level of initiation, in a dose-dependent fashion. The present studies confirm the existence of translational control in skeletal muscle and suggest that it operates at the embryonic to adult transition. A model of muscle cell differentiation, based upon transcriptional control at the myoblast level, followed by translational regulation at the level of the postmitotic myoblast and/or myotube, is proposed.

A genetic pathway for regulation of tra-2 translation

Development ◽  
1997 ◽  
Vol 124 (3) ◽  
pp. 749-758 ◽  
Author(s):  
E.B. Goodwin ◽  
K. Hofstra ◽  
C.A. Hurney ◽  
S. Mango ◽  
J. Kimble
Keyword(s):  
Translational Control ◽  
Direct Repeat ◽  
Postembryonic Development ◽  
Early Embryo ◽  
Translational Repression ◽  
Female Development ◽  
Normal Pattern ◽  
Repeat Elements ◽  
Translation Repression ◽  
Translational Level

In Caenorhabditis elegans, the tra-2 sex-determining gene is regulated at the translational level by two 28 nt direct repeat elements (DREs) located in its 3′ untranslated region (3′UTR). DRF is a factor that binds the DREs and may be a trans-acting translational regulator of tra-2. Here we identify two genes that are required for the normal pattern of translational control. A newly identified gene, called laf-1, is required for translational repression by the tra-2 3′UTR. In addition, the sex-determining gene, tra-3, appears to promote female development by freeing tra-2 from laf-1 repression. Finally, we show that DRF activity correlates with translational repression of tra-2 during development and that tra-3 regulates DRF activity. We suggest that tra-3 may promote female development by releasing tra-2 from translation repression by laf-1 and that translational control is important for proper sex determination--both in the early embryo and during postembryonic development.

scholarly journals Neutrophil azurophilic granule glycoproteins are distinctively decorated by atypical pauci- and phosphomannose glycans

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Karli R. Reiding ◽  
Yu-Hsien Lin ◽  
Floris P. J. van Alphen ◽  
Alexander B. Meijer ◽  
Albert J. R. Heck
Keyword(s):  
Immune System ◽  
Autoimmune Disease ◽  
Secreted Proteins ◽  
Receptor Interaction ◽  
Post Translational Modification ◽  
Site Specific ◽  
Healthy Donors ◽  
Asymmetric Hybrid ◽  
Membrane Bound ◽  
High Mannose

AbstractWhile neutrophils are critical first-responders of the immune system, they also cause tissue damage and act in a variety of autoimmune diseases. Many neutrophil proteins are N-glycosylated, a post-translational modification that may affect, among others, enzymatic activity, receptor interaction, and protein backbone accessibility. So far, a handful neutrophil proteins were reported to be decorated with atypical small glycans (paucimannose and smaller) and phosphomannosylated glycans. To elucidate the occurrence of these atypical glycoforms across the neutrophil proteome, we performed LC-MS/MS-based (glyco)proteomics of pooled neutrophils from healthy donors, obtaining site-specific N-glycan characterisation of >200 glycoproteins. We found that glycoproteins that are typically membrane-bound to be mostly decorated with high-mannose/complex N-glycans, while secreted proteins mainly harboured complex N-glycans. In contrast, proteins inferred to originate from azurophilic granules carried distinct and abundant paucimannosylation, asymmetric/hybrid glycans, and glycan phosphomannosylation. As these same proteins are often autoantigenic, uncovering their atypical glycosylation characteristics is an important step towards understanding autoimmune disease and improving treatment.

Circadian Synthesis of Light-Harvesting-Chlorophyll-Proteins in Euglena gracilis Is under Translational Control

1998 ◽  
Vol 53 (11-12) ◽  
pp. 1017-1026 ◽  
Author(s):  
A. Kiinne ◽  
E. Pistorius ◽  
K. Kloppstech ◽  
E. de Groot
Keyword(s):  
Euglena Gracilis ◽  
Translational Control ◽  
Light Harvesting ◽  
Higher Plants ◽  
Mrna Levels ◽  
Stationary Concentration ◽  
The Family ◽  
Molecular Masses ◽  
Lhcp Ii ◽  
Translational Level

Abstract Two proteins with apparent molecular masses of 17 and 24 kD that are synthesized in a circadian manner in the phytoflagellate Euglena gracilis, were recognized as proteins belong­ing to the family of light-harvesting-chlorophyll-proteins (LHCPs) of class I (17 kD) and of class II (24 kD). Identification was achieved by N-terminal sequencing of the proteins isolated from two-dimensional polyacrylamide gels and by detection with an anti-LHCP II se­rum. While it was found that the total amount of LHCPs remains almost constant, when Euglena is grown under diurnal conditions (12 h light and 12 h dark), we could show that the amount of newly synthesized 17 and 24 kD proteins varies about 20-fold with a maximum of synthesis in the light phase. In contrast, the analysis of the mRNA levels at different times revealed only minor differences in the stationary concentration of the LHCP specific mRNA, indicating that the control of LHCP synthesis is at the translational level. Principally, the same finding was obtained using inhibitors of transcription. Thus, it is concluded that the expression of LHCPs in Euglena gracilis in contrast to that of higher plants is primarily regulated at the translational level.

scholarly journals Defective glycosylation of erythrocyte membrane glycoconjugates in a variant of congenital dyserythropoietic anemia type II: association of low level of membrane-bound form of galactosyltransferase

Blood ◽  
1989 ◽  
Vol 73 (5) ◽  
pp. 1331-1339 ◽  
Author(s):  
MN Fukuda ◽  
KA Masri ◽  
A Dell ◽  
EJ Thonar ◽  
G Klier ◽  
...  
Keyword(s):  
Genetic Disease ◽  
Fast Atom Bombardment ◽  
Mass Spectrometry Analysis ◽  
Type Ii ◽  
Congenital Dyserythropoietic Anemia ◽  
Spectrometry Analysis ◽  
Microsomal Membranes ◽  
Cda Ii ◽  
Membrane Bound ◽  
High Mannose

Abstract Congenital dyserythropoietic anemia type II (CDA II) or HEMPAS is a genetic disease caused by plasma membrane abnormality. The enzymic defect of HEMPAS has been suggested to be the lowered activity of N- acetylglucosaminyltransferase II, resulting in lack of polylactosamine formation on proteins and leading to accumulation of polylactosaminyl lipids. In contrast to typical HEMPAS cases, cell-surface labeling of the erythrocytes of a HEMPAS variant G.K. showed an absence of polylactosamines either on proteins or on lipids. Fast-atom bombardment mass spectrometry analysis of G.K.'s erythrocyte glycopeptides detected a series of high mannose-type oligosaccharides, which were not detected in erythrocyte N-glycans of normal cells or of other HEMPAS cases: The former contains polylactosaminoglycans and the latter contains hybrid- type oligosaccharides. Keratansulfate (sulfated polylactosamines) in this patient's serum was abnormally low. The galactosyltransferase activity in microsomal membranes prepared from G.K.'s mononucleated cells was 24% of the normal level, whereas this enzyme activity in G.K.'s serum was comparatively higher than normal. Western blotting of G.K.'s membranes using antigalactosyltransferase antibodies showed that G.K. has reduced amounts of this enzyme present. The results collectively suggest that variant G.K. is defective in polylactosamine synthesis owing to the decreased quantity of the membrane-bound form of galactosyltransferase.

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