In vitro biosynthesis of the lysosomal cathepsin H

A lysosomal thiol protease cathepsin H has been synthesized in vitro and shown to undergo co-translational segregation into the lumen of microsomal vesicles. Using cell-free synthesis, a 36 K Da cathepsin H was found to be synthesized exclusively on membrane-bound polysomes. When the microsomal membrane were present during translation, a glycosylated 41 K Da proenzyme appeared in the microsomal lumen. This proenzyme was converted to a 34 K Da protein by endoglycosidase H treatment. These results suggest that the nascent chain of cathepsin H has a transient N-terminal prepropeptide.     

Transport of an immunoglobulin light chain fragment across the endoplasmic reticulum does not require an amino terminal variable region: Implications for the signal hypothesis

The co-translational processing of the kappa light chain constant region fragment was investigated in a Krebs ascites cell-free system. The fragment which is devoid of the entire variable region was initially synthesized as a precursor which contains a signal (pre) peptide. Addition of microsomal membranes to the reaction mixtures resulted in the synthesis of a 11,600 dalton product which was sequestered in the vesicles. Amino acid sequence analysis indicated that the pre-peptide had been removed. The data show that despite the absence of the amino terminal variable region, the fragment was processed and translocated across the endoplasmic reticulum. The data suggest that the presumed recognition regions necessary for translocating eukaryotic secretory proteins across the endoplasmic reticulum do not reside in the amino terminal region near the signal peptide.     

Residues flanking the COOH-terminal C-region of a model eukaryotic signal peptide influence the site of its cleavage by signal peptidase and the extent of coupling of its co-translational translocation and proteolytic processing in vitro

The polar, COOH-terminal c-region of signal peptides has been considered to be most important for influencing the efficiency and fidelity of signal peptidase cleavage while the hydrophobic core or h-region appears indispensable for initiating translocation. To identify structural features of residues flanking the c-region that influence the fidelity and efficiency of signal peptidase cleavage as well as co-translational translocation, we introduced six amino acid substitutions into the COOH terminus of the hydrophobic core and seven substitutions at the NH2 terminus of the mature region (the +1 position) of a model eukaryotic preprotein-human pre(delta pro)apoA-II. This preprotein contains several potential sites for signal peptidase cleavage. The functional consequences of these mutations were assayed using an in vitro co-translational translocation/processing system and by post-translational cleavage with purified, detergent-solubilized, hen oviduct signal peptidase. The efficiency of translocation could be correlated with the hydrophobic character of the residue introduced at the COOH terminus of the h-region. Some h/c boundary mutants underwent co-translational translocation across the microsomal membrane with only minimal cleavage yet they were cleaved post-translationally by hen oviduct signal peptidase more efficiently than other mutants which exhibited a high degree of coupling of co-translational translocation and cleavage. These data suggest that features at the COOH terminus of the h-domain can influence "presentation" of the cleavage site to signal peptidase. The +1 residue substitutions had minor effects on the extent of co-translational translocation and processing. However, these +1, as well as h/c boundary mutations, had dramatic effects on the site of cleavage chosen by signal peptidase, indicating that residues flanking the c-region of this prototypic eukaryotic signal peptide can affect the fidelity of its proteolytic processing. The site(s) selected by canine microsomal and purified hen oviduct signal peptidase were very similar, suggesting that "intrinsic" structural features of this prepeptide can influence the selectivity of eukaryotic signal peptidase cleavage, independent of the microsomal membrane and associated translocation apparatus.     

Cotranslational glycosylation of proteins in systems depleted of protein disulphide isomerase.

The role of protein disulphide isomerase (PDI) and other resident proteins of the endoplasmic reticulum (ER) lumen in co- and post-translational modification of secretory proteins has been studied in experiments on translation in vitro. We have devised procedures for extracting the lumenal content proteins of dog pancreas microsomal vesicles by alkaline buffer, or detergent washing, and for reconstitution of the depleted membrane fraction. When microsomal membranes are depleted of content by washing at pH 9.1, they are able to co-translationally glycosylate human interferon-gamma (IFN-gamma) and yeast pro-alpha-factor and the products appear to be identical to those produced by control microsomes. However, when microsomal membranes are depleted of content by washing with saponin they are still able to co-translationally translocate and glycosylate human IFN-gamma, but the products were of higher apparent Mr than those generated by control microsomes. When saponin-washed microsomal membranes were reconstituted with homogeneous protein disulphide isomerase (PDI), the generated vesicles gave the same pattern of co-translationally glycosylated IFN-gamma as saponin-washed microsomal membranes lacking PDI. These results are discussed in relation to the roles of resident ER proteins in co-translational modification; they suggest that PDI is not an essential component of the machinery of co-translational N-glycosylation, but that detergent washing may inactivate or remove some ER glycosidases.     

Co-translational modification of nascent immunoglobulin heavy and light chains

We have investigated the in vivo co-translational covalent modification of nascent immunoglobulin heavy and light chains. Nascent polypeptides were separated from completed polypeptides by ion-exchange chromatography of solubilized ribosomes on QAE-Sephadex. First, we have demonstrated that MPC 11 nascent heavy chains are quantitatively glycosylated very soon after the asparaginyl acceptor site passes through the membrane into the cisterna of the rough endoplasmic reticulum. Nonglycosylated completed heavy chains of various classes cannot be glycosylated after release from the ribosome, due either to rapid intramolecular folding and/or intermolecular assembly, which cause the acceptor site to become unavailable for the glycosylation enzyme. Second, we have shown that the formation of the correct intrachain disulfide loop within the first light chain domain occurs rapidly and quantitatively as soon as the appropriate cysteine residues of the nascent light chain pass through the membrane into the cisterna of the endoplasmic reticulum. The intrachain disulfide loop in the second or constant region domain of the light chain is not formed on nascent chains, because one of the cysteine residues involved in this disulfide bond does not pass through the endoplasmic reticulum membrane prior to chain completion and release from the ribosome. Third, we have demonstrated that some of the initial covalent assembly (formation of interchain disulfide bonds) occurs on nascent heavy chains prior to their release from the ribosome. The results are consistent with the pathway of covalent assembly of the cell line, in that completed light chains are assembled onto nascent heavy chains in MPC 11 cells (IgG₂b), where a heavy-light half molecule is the major initial covalent intermediate; and completed heavy chains are assembled onto nascent heavy chains in MOPC 21 cells (IgG₁), where a heavy chain dimer is the major initial disulfide linked intermediate.     

Cell-free processing and segregation of insulin precursors

The biosynthesis, segregation, and processing of preproinsulin (116 amino acids) was investigated to determine the mechanism(s) by which it is translocated across the endoplasmic reticulum membrane. Islet mRNA was translated in the wheat germ cell-free system, and at various times during preproinsulin synthesis, puromycin was added, followed by addition of microsomal membranes. Neither processing of preproinsulin nor translocation of proinsulin into microsomal membranes occurred in the presence of puromycin. Synchronization of preproinsulin translation by addition of 7-methylguanosine 5'-phosphate enabled the timing of preproinsulin synthesis and proinsulin (91 amino acids) segregation into microsomal membranes to be determined. Membrane binding occurs when about 60 amino acids have been polymerized, i.e. prior to the completion of the polypeptide chain. The binding of signal recognition particle to the nascent signal is demonstrated to be an absolute requirement for translocation and processing of preproinsulin. The results indicate that segregation and processing of preproinsulin are co-translational events; no evidence for a post-translational mechanism was found. Furthermore, this work, together with similar studies, suggests that presecretory polypeptides must be synthesized as part of a precursor with a minimum size of 60-80 amino acids in order to effect membrane binding and translocation of the polypeptide chain within the intracisternal space of the endoplasmic reticulum.     

Co-translational protein targeting catalyzed by the Escherichia coli signal recognition particle and its receptor.

The Ffh-4.5S ribonucleoprotein particle (RNP) and FtsY from Escherichia coli are homologous to essential components of the mammalian signal recognition particle (SRP) and SRP receptor, respectively. The ability of these E. coli components to function in a bona fide co-translational targeting pathway remains unclear. Here we demonstrate that the Ffh-4.5S RNP and FtsY can efficiently replace their mammalian counterparts in targeting nascent secretory proteins to microsomal membranes in vitro. Targeting in the heterologous system requires a hydrophobic signal sequence, utilizes GTP and, moreover, occurs co-translationally. Unlike mammalian SRP, however, the Ffh-4.5S RNP is unable to arrest translational elongation, which results in a narrow time window for the ribosome nascent chain to interact productively with the membrane-bound translocation machinery. The highly negatively charged N-terminal domain of FtsY, which is a conserved feature among prokaryotic SRP receptor homologs, is important for translocation and acts to localize the protein to the membrane. Our data illustrate the extreme functional conservation between prokaryotic and eukaryotic SRP and SRP receptors and suggest that the basic mechanism of co-translational protein targeting is conserved between bacteria and mammals.     

Calmodulin-activated protein kinase activity of adipocyte microsomes

A calmodulin-activated phosphorylation activity was identified in microsomal (endoplasmic reticulum) preparations from rat adipocytes. Activity was not detected in mitochondrial or plasma membrane fractions. Although the phosphorylation of several proteins was enhanced by addition of calmodulin, the major calmodulin-sensitive protein had a molecular weight of 54,000. A series of experiments were conducted to determine if the microsomal phosphorylation was either calmodulin-containing phosphorylase kinase or calmodulindependent myosin light chain kinase. The phosphorylation of the 54,000 Dalton band in microsomal preparations was 1) not significantly reduced by potential competing protein substrates, e.g. actomyosin or phosphorylase b, 2) nearly equally well phosphorlyated at pH 8.6 or pH 7.0, unlike actomyosin or phosphorylase b, and 3) not increased by addition of phosphorylase kinase or myosin light chain kinase. The results demonstrate that this microsomal calmodulinactivated phosphorylation is catalysed by a protein kinase distinct from phosphorylase kinase or myosin light chain kinase.     

Accumulation of immunoglobulin messenger ribonucleic acid in immunized mouse spleen.

We have measured the concentration of mRNAs coding for immunoglobulins, k and lambda type light chains and gamma 1 type heavy chain, in mouse spleen cells activated by bacterial lipopolysaccharide or sheep red blood cells. These mRNAs were quantitated by hybridization to radioactive DNA complementary to highly purified immunoglobulin mRNAs from mouse myelomas. In the lipopolysaccharide-stimulated spleen cells, only light chain mRNA accumulated, whereas gamma 1 type heavy chain mRNA remained unvaried. The light chain mRNA concentration also increased in purified bone-marrow-derived lymphocytes. The lipopolysaccharide-induced light chain mRNA was similar to light chain mRNAs purified from myelomas. The accumulation and disappearance of light chain mRNA in bone-marrow-derived lymphocytes coincide with the kinetics of synthesis of immunoglobulin M which is the major species induced by lipopolysaccharide. In sheep red blood cell stimulated spleen, the specific accumulation of k type light chain and gamma 1 type heavy chain mRNAs parallels immunoglobulin G synthesis. These results seem to indicate that the increment of immunoglobulin mRNA concentration in bone-marrow-derived lymphocytes is important for induction of immunoglobulin synthesis.     

Post-translational transport of proteins into microsomal membranes of Candida maltosa.

We have isolated from the yeast Candida maltosa microsomal membranes that are active in the translocation of proteins synthesized in cell-free systems derived from C. maltosa, Saccharomyces cerevisiae or wheat germ. Translocation and core glycosylation of prepro-alpha-factor, a secretory protein, were observed with yeast microsomes added during or after translation. The signal peptide is cleaved off. Cytochrome P-450 from C. maltosa, the first integral membrane protein studied in a yeast system, is also inserted both co- and post-translationally into Candida microsomal membranes. Its insertion into canine microsomes occurs efficiently only in a co-translational manner and is dependent on the function of the signal recognition particle.     

Characterization of light chain and light chain constant region fragment mRNAs in MPC 11 mouse myeloma cells and variants

Cultured MPC 11 mouse myeloma cells synthesize not only γ_2b heavy and κ light chains but also a carboxyl terminal (constant region) fragment of κ light chain. In vitro translational analysis of total cytoplasmic and microsomal RNA indicates that these cells contain RNA which directs synthesis of both a light chain precursor and a light chain fragment precursor. Variant clones which do not synthesize either heavy or light chains continue to synthesize the light chain fragment. One such “nonproducing” variant was studied in detail. It does not contain translatable mRNA for the intact light chain but does contain RNA which is translated into the light chain fragment precursor. Nucleic acid hybridization analysis with a cDNA probe specific for the constant region of κ light chains revealed that microsomal RNA from the wild-type cell contains both a 14S and a 10S species of κ specific RNA, whereas the variant contains only the 10S species. Translational analysis of these same RNAs indicates that the 14S species codes for the light chain precursor, while the 10S RNA codes for the light chain fragment precursor.     

甲型H1N1流感病毒血凝素单克隆抗体轻链和重链可变区基因的巢式PCR扩增及序列分析

目的：采用巢式PCR对甲型H1N1流感病毒血凝素单克隆抗体的轻链和重链基因进行扩增,对获得的基因进行序列分析,并找出克隆鼠Igκ轻链和重链可变区基因的通用方法。方法：设计22对扩增鼠Igκ轻链可变区和重链可变区基因的引物,对6株鼠抗人甲型H1N1流感病毒血凝素单克隆抗体的轻链和重链可变区基因进行克隆并测序,与NCBI公布的鼠免疫球蛋白序列比对分析。结果：巢式PCR方法可以有效避免单克隆抗体克隆过程的假基因,并且得到的单克隆抗体的氨基酸序列均符合鼠免疫球蛋白可变区特征。结论：建立了克隆鼠免疫球蛋白轻链和重链可变区基因的通用方法,为后期克隆鼠源性单克隆抗体的可变区基因提供了基础,并为研究甲型H1N1流感病毒血凝素与抗体的结合位点提供了实验数据。     

Immunocytochemical identification and localization of immunoglobulin A within Paneth cells of the rat small intestine.

Light microscopic immunocytochemistry was used to identify Paneth cells by their lysozyme content and to detect immunoglobulin antigens within a subpopulation of these cells. Antisera specific for the heavy chains of rat or human immunoglobulin A and for immunoglobulin light chain antigens produced specific staining of rat Paneth cells. The distribution of immunoglobulin staining varied between adjacent Paneth cells in the same crypt and between Paneth cells in adjacent crypts, as well as between Paneth cell populations of different animals. No staining of rat Paneth cells was detected using antisera specific for the heavy chain of immunoglobulins G or M. The specific staining of Paneth cells for immunoglobulin A and light chain antigens was blocked by absorption of each antiserum with its respective purified antigen. Absorption of these antisera with purified rat lysozyme did not affect staining and thereby eliminated the possibility of immunologic cross-reactivity between lysozyme and immunoglobulin antigens. It is suggested, in light of current concepts of Paneth cell function, that the immunoglobulin staining of Paneth cells may reflect their ability to phagocytize immunoglobulin A-coated microorganisms or immune complexes containing immunoglobulin A.     

Dog pancreatic microsomal-membrane polypeptides analysed by two-dimensional gel electrophoresis.

The two-dimensional polyacrylamide-gel electrophoresis technique of O'Farrell [(1975) J. Biol. Chem 250, 4007-4021] was applied to resolve and analyse the polypeptide composition of dog pancreatic rough microsomal membranes, which were shown to be active in co-translational processing of preprolactin synthesized from pituitary mRNA in a translation system in vitro. About 100 polypeptides are resolved. Treatment of rough microsomal membranes with EDTA and high KCl concentration yielded membranes stripped of their ribosomes with retention of activity for translocation and processing. Stripped microsomal membranes showed a selective concentration of approximately 25 polypeptides in the membranes when analysed by two-dimensional polyacrylamide-gel electrophoresis. The two-dimensional electrophoretic profile was catalogued into polypeptides that are glycoproteins, those that contain free thiol groups disposed at the cytosolic surface of microsomal vesicles and those that are of secretory origin but have been entrapped in the microsomal preparation. Several secretory components, including amylase, procarboxypeptidases, lipase and anionic trypsinogen, were tentatively identified among the microsomal polypeptides. The rough and stripped microsomal membranes from dog pancreas show a characteristic set of seven major acidic polypeptides, which are also identifiable in microsomal-membrane preparations isolated from dog liver and rat liver. One of these polypeptides was identified as protein disulphide-isomerase (EC 5.3.4.1).     

The vectorial release of nascent immunoglobulin peptides

A microsomal preparation from a mouse plasmacytoma, MOPC 47A, that secretes immunoglobulin A was used to study the release of nascent immunoglobulin peptides in vitro. Nascent chains were released with puromycin and characterized with specific antiserum against the immunoglobulin product of the tumour. When the tissue had been prelabelled with [(3)H]leucine the experiments were complicated by the large background of completed radioactive polypeptides in the microsomal preparation. Up to one-third of the released radioactivity in the microsomal preparation could be recognized as immunoglobulin. With [(3)H]-puromycin as the radioactive label, however, the results are much easier to interpret, although the proportion of released radioactivity that can be identified as immunoglobulin is lower (up to one-tenth). Both types of experiment demonstrate that all of the recognizable nascent immunoglobulin chains remain in association with the microsomal vesicles after release from the ribosomes.     

PAK4 phosphorylates myosin regulatory light chain and contributes to Fcγ receptor-mediated phagocytosis

Phagocytosis of immunoglobulin G-opsonised particles takes place via Fcγ receptor ligation, leading to uptake through an actin-dependent mechanism. Myosin regulatory light chains have previously been reported to control contractility during uptake through the Fcγ receptor. In this study, we show that p21-activated kinase 4 contributes to Fcγ receptor-mediated uptake downstream of actin cup formation by regulating phosphorylation of myosin regulatory light chain. siRNA-mediated knockdown of p21-activated kinase 4 leads to reduced myosin regulatory light chain phosphorylation at Serine 19, with a corresponding reduction in phospho-myosin regulatory right chain localised to bound immunoglobulin G-opsonised red blood cells. p21-activated kinase 4 phosphorylates myosin light chain 9 at Serine 19 in vitro and RNA interference against myosin light chain 9 implicates this isoform, but not myosin light chain 12A or 12B, in Fcγ receptor-mediated uptake. Taken together, these data indicate that p21-activated kinase 4 regulates regulatory myosin light chain phosphorylation and myosin contractility during FcγR-mediated phagocytosis.     

Light chain gene conversion continues at high rate in an ALV-induced cell line.

We have analyzed immunoglobulin light chain sequences from avian leukosis virus (ALV) induced bursal and metastatic tumors and from cell lines derived from these tumors. Sequence data presented demonstrate that ALV-induced tumors and one cell line (DT40) derived therefrom continue to diversify their light chain genes outside of the bursal environment. Diversification within these tumor cells seems to occur by gene conversion events comparable with those observed in bursal B cells. Sequence analysis of spontaneously arising surface immunoglobulin negative subclones of the DT40 cell line revealed frameshifts within the rearranged light chain genes which most likely resulted from non-functional recombination events. Superimposed gene conversion events can repair these frameshifts leading to re-expression of surface immunoglobulin.