Glomerular and tubular handling of differently charged human immunoglobulin light chains by the rat kidney

1988 ◽  
Vol 74 (6) ◽  
pp. 639-644 ◽  
Author(s):  
Christine Baylis ◽  
James Falconer-Smith ◽  
Brian Ross
Keyword(s):  
Light Chain ◽  
Rat Kidney ◽  
Tubular Reabsorption ◽  
High Rate ◽  
Light Chains ◽  
Renal Handling ◽  
Charge Dependence ◽  
Intact Kidney

1. To examine the effect of charge and molecular size on the renal handling of immunoglobulin light chain, three different human light chains were purified, iodinated and characterized with regard to molecular weight and isoelectric point. The molecular weights were similar, whereas the isoelectric points ranged from markedly cationic to markedly anionic. 2. Renal handling of the light chains was determined in vivo in the Munich–Wistar rat and in vitro in the isolated perfused rat kidney. 3. Significant restriction to the transglomerular passage of all three light chains was evident in the intact kidney, with the most anionic light chain being restricted most while the cationic light chain was restricted least. The charge dependence of filtration of these naturally occurring proteins was, however, much less pronounced than has previously been reported for the synthetic dextrans. 4. Tubular reabsorption of the three light chains was quite variable in the intact kidney and did not appear to be related to molecular charge. In the isolated perfused kidney, once account had been taken of the abnormally high rate of filtration of these proteins, the reabsorption of the three light chains was strikingly similar to that seen in vivo. 5. With these three purified human light chains no marked influence of charge (pI) has been demonstrated in vivo or in vitro, for glomerular restriction or renal tubular reabsorption. Some other properties of human myeloma light chains may determine their renal handling and nephrotoxicity.

An Essential Role of the Factor VIII Light Chain in Facilitating Heavy Chain Secretion.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4034-4034
Author(s):  
Lingxia Chen ◽  
Juan Li ◽  
Hui Lu ◽  
Haiyan Jiang ◽  
Rita Sarkar ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Hek293 Cells ◽  
Light Chains ◽  
Trans Splicing ◽  
In Cis

Abstract Blood coagulation Factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy and light chain. Both in vitro and in vivo studies have demonstrated that these chains can be expressed independently. The expressed heavy and light chains can reassociate with recovery of biological activities. These observations have been particularly useful in a gene therapy setting since vector packaging capacity for adeno-associated virus (AAV) is a limiting factor. However, it has been demonstrated that the FVIII heavy chain is expressed ~10–100-fold less efficiently compared to the light chain when expressed independently. Previously the FVIII F309S mutation in the context of B-domainless FVIII (FVIII-BDD) and enhanced glycosylations within the B-domain have been shown to improve factor VIII expression and secretion. However, our in vitro studies indicate that these improvements in secretion were not retained when expressing the heavy chain alone with the same modifications. Other sequences, possibly in the light chain, may facilitate secretion. To investigate this further, we designed an intein trans-splicing strategy to control the addition of light chain to the heavy chain before secretion. Using HEK293 cells, we cotransfected seperate intein light chain and intein heavy chain plasmids and compared results to single plasmid transfected cells. 48 hours post-transfection, FVIII-specific ELISA results demonstrated that cotransfection of intein heavy chain and intein light chain had a significant influence on total heavy chain secretion compared to intein heavy chain expression alone. The co-transfected intein heavy chain and intein light chain were efficiently ligated together yielding a biologically active single chain FVIII derivative as demonstrated by clotting assays and Western blot analysis. Therefore, heavy chain secretion was directly enhanced by the attachment of the light chain to the C-terminus of the heavy chain. A similar phenomenon was not found when heavy and light chains were simply co-expressed in the same cell. It suggested that light chain functioned in cis. Hydrodynamic injection of plasmids with intein heavy chain and intein light chain into hemophilia A mice led to a much higher level of FVIII secretion. The amount of functional FVIII expression reached 3–6 units/ml at peak level. In the absence of intein light chain, FVIII heavy chain secretion was approximately 100 fold less efficient in vivo. To map the key elements of FVIII light in helping FVIII secretion, we made deletion variants in the light chain. These mutants had a dominant negative effect in reducing FVIII and FVIII heavy chain secretion while increasing the level of intracellular FVIII accumulation. Collectively our results are consistent with the conclusion that the FVIII light chain plays a critical role in facilitating heavy chain secretion in cis; probably through helping FVIII heavy chain maintain correct configuration and folding. The strategy to manipulate FVIII light chain addition through intein mediated trans-splicing reaction may also be explored for human gene therapy.

scholarly journals ERdj3, a Stress-inducible Endoplasmic Reticulum DnaJ Homologue, Serves as a CoFactor for BiP's Interactions with Unfolded Substrates

2005 ◽  
Vol 16 (1) ◽  
pp. 40-50 ◽  
Author(s):  
Ying Shen ◽  
Linda M. Hendershot
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Folding ◽  
Atpase Activity ◽  
Light Chain ◽  
Light Chains ◽  
Nonpermissive Temperature ◽  
Protein Substrates ◽  
Unfolded Protein

We recently identified ERdj3 as a component of unassembled immunoglobulin (Ig) heavy chain:BiP complexes. ERdj3 also associates with a number of other protein substrates, including unfolded light chains, a nonsecreted Ig light chain mutant, and the VSV-G ts045 mutant at the nonpermissive temperature. We produced an ERdj3 mutant that was unable to stimulate BiP's ATPase activity in vitro or to bind BiP in vivo. This mutant retained the ability to interact with unfolded protein substrates, suggesting that ERdj3 binds directly to proteins instead of via interactions with BiP. BiP remained bound to unfolded light chains longer than ERdj3, which interacted with unfolded light chains initially, but quickly disassociated before protein folding was completed. This suggests that ERdj3 may bind first to substrates and serve to inhibit protein aggregation until BiP joins the complex, whereas BiP remains bound until folding is complete. Moreover, our findings support a model where interactions with BiP help trigger the release of ERdj3 from the substrate:BiP complex.

scholarly journals Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma.

1975 ◽  
Vol 67 (3) ◽  
pp. 835-851 ◽  
Author(s):  
G Blobel ◽  
B Dobberstein
Keyword(s):  
Light Chain ◽  
Light Chains ◽  
Discussion Section ◽  
Heterologous System ◽  
In Vitro Processing ◽  
Nascent Chain ◽  
Detergent Treatment ◽  
Membrane Bound

Fractionation of MOPC 41 DL-1 tumors revealed that the mRNA for the light chain of immunoglobulin is localized exclusively in membrane-bound ribosomes. It was shown that the translation product of isolated light chain mRNA in a heterologous protein-synthesizing system in vitro is larger than the authentic secreted light chain; this confirms similar results from several laboratories. The synthesis in vitro of a precursor protein of the light chain is not an artifact of translation in a heterologous system, because it was shown that detached polysomes, isolated from detergent-treated rough microsomes, not only contain nascent light chains which have already been proteolytically processed in vivo but also contain unprocessed nascent light chains. In vitro completion of these nascent light chains thus resulted in the synthesis of some chains having the same mol wt as the authentic secreted light chains, because of completion of in vivo proteolytically processed chains and of other chains which, due to the completion of unprocessed chains, have the same mol wt as the precursor of the light chain. In contrast, completion of the nascent light chains contained in rough microsomes resulted in the synthesis of only processed light chains. Taken together, these results indicate that the processing activity is present in isolated rough microsomes, that it is localized in the membrane moiety of rough microsomes, and, therefore, that it was most likely solubilized during detergent treatment used for the isolation of detached polysomes. Furthermore, these results established that processing in vivo takes place before completion of the nascent chain. The data also indicate that in vitro processing of nascent chains by rough microsomes is dependent on ribosome binding to the membrane. If the latter process is interfered with by aurintricarboxylic acid, rough microsomes also synthesize some unprocessed chains. The data presented in this paper have been interpreted in the light of a recently proposed hypothesis. This hypothesis, referred to as the signal hypothesis, is described in greater detail in the Discussion section.

scholarly journals IgG1 Thioether Bond Formation in Vivo

2013 ◽  
Vol 288 (23) ◽  
pp. 16371-16382 ◽  
Author(s):  
Qingchun Zhang ◽  
Matthew R. Schenauer ◽  
John D. McCarter ◽  
Gregory C. Flynn
Keyword(s):  
Light Chain ◽  
Reaction Rate ◽  
Human Subjects ◽  
Light Chains ◽  
Chain Reaction ◽  
Healthy Human ◽  
Conversion Rates ◽  
Healthy Human Subjects

During either production or storage, the LC214-HC220 disulfide in therapeutic antibodies can convert to a thioether bond. Here we report that a thioether forms at the same position on antibodies in vivo. An IgG1κ therapeutic antibody dosed in humans formed a thioether at this position at a rate of about 0.1%/day while circulating in blood. Thioether modifications were also found at this position in endogenous antibodies isolated from healthy human subjects, at levels consistent with this conversion rate. For both endogenous antibodies and recombinant antibodies studied in vivo, thioether conversion rates were faster for IgG1 antibodies containing λ light chains than those containing κ light chains. These light chain reaction rate differences were replicated in vitro. Additional mechanistic studies showed that base-catalyzed thioether formation through the light chain dehydrogenation was more preferred on antibodies with λ light chains, which may help explain the observed reaction rate differences.

scholarly journals BiP and Immunoglobulin Light Chain Cooperate to Control the Folding of Heavy Chain and Ensure the Fidelity of Immunoglobulin Assembly

1999 ◽  
Vol 10 (7) ◽  
pp. 2209-2219 ◽  
Author(s):  
Young-Kwang Lee ◽  
Joseph W. Brewer ◽  
Rachel Hellman ◽  
Linda M. Hendershot
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Protein Complexes ◽  
Critical Role ◽  
Light Chains ◽  
Heavy Chains ◽  
Chain Complexes ◽  
Chain Synthesis

The immunoglobulin (Ig) molecule is composed of two identical heavy chains and two identical light chains (H2L2). Transport of this heteromeric complex is dependent on the correct assembly of the component parts, which is controlled, in part, by the association of incompletely assembled Ig heavy chains with the endoplasmic reticulum (ER) chaperone, BiP. Although other heavy chain-constant domains interact transiently with BiP, in the absence of light chain synthesis, BiP binds stably to the first constant domain (CH1) of the heavy chain, causing it to be retained in the ER. Using a simplified two-domain Ig heavy chain (VH-CH1), we have determined why BiP remains bound to free heavy chains and how light chains facilitate their transport. We found that in the absence of light chain expression, the CH1 domain neither folds nor forms its intradomain disulfide bond and therefore remains a substrate for BiP. In vivo, light chains are required to facilitate both the folding of the CH1 domain and the release of BiP. In contrast, the addition of ATP to isolated BiP–heavy chain complexes in vitro causes the release of BiP and allows the CH1 domain to fold in the absence of light chains. Therefore, light chains are not intrinsically essential for CH1 domain folding, but play a critical role in removing BiP from the CH1 domain, thereby allowing it to fold and Ig assembly to proceed. These data suggest that the assembly of multimeric protein complexes in the ER is not strictly dependent on the proper folding of individual subunits; rather, assembly can drive the complete folding of protein subunits.

Characteristics of Renal Handling of Human Immunoglobulin Light Chain by the Perfused Rat Kidney

1979 ◽  
Vol 57 (1) ◽  
pp. 113-120 ◽  
Author(s):  
J. F. Falconer Smith ◽  
R. I. Van Hegan ◽  
M. P. Esnouf ◽  
B. D. Ross
Keyword(s):  
Light Chain ◽  
Prolonged Exposure ◽  
Rat Kidney ◽  
Human Immunoglobulin ◽  
Light Chains ◽  
Immunoglobulin Light Chain ◽  
Renal Handling ◽  
Perfused Rat Kidney ◽  
Renal Tubular ◽  
Sodium And Potassium

1. The renal handling of purified human immunoglobulin light chain has been studied with an isolated perfused rat kidney preparation. 2. Human immunoglobulin light chain was freely filtered and largely reabsorbed. Fractional reabsorption was characteristic for each of four light chains and varied between 56% and 86%. No renal tubular maximum for human light chain was obtained. 3. Light chains at concentrations up to 10 times those seen in human myeloma were without effect on glomerular filtration rate or sodium and potassium reabsorption in experiments lasting up to 2h. 4. Filtered and reabsorbed light chain returned ultimately to the perfusion medium, indicating a unique property of the tubular handling of this protein. None of the inhibitors tested (ouabain, frusemide, acetazolamide, probenicid) influenced light chain reabsorption. 5. The results are taken to indicate that light chain reaches the site of the transport enzyme, Na+, K+−dependent ATPase, at concentrations which vary with the nature of the light chain. This may provide a mechanism for renal damage in patients with myeloma, after prolonged exposure.

scholarly journals STEM-26. SMALL MOLECULE DRUG CBL0137 INHIBITS THE FACT COMPLEX AND SENSITIZES GLIOBLASTOMA CANCER STEM CELLS TO RADIOTHERAPY

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii201-ii202
Author(s):  
Miranda Tallman ◽  
Abigail Zalenski ◽  
Amanda Deighen ◽  
Morgan Schrock ◽  
Sherry Mortach ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
High Rate ◽  
Orthotopic Model ◽  
In Vivo Models ◽  
Specific Cytotoxicity ◽  
Treatment Paradigm ◽  
Cancer Agent

Abstract Glioblastoma (GBM) is a malignant brain tumor with nearly universal recurrence. GBM cancer stem cells (CSCs), a subpopulation of radio- and chemo-resistant cancer cells capable of self-renewal, contribute to the high rate of recurrence. The anti-cancer agent, CBL0137, inhibits the FACT (facilitates chromatin transcription) complex leading to cancer cell specific cytotoxicity. Here, we show that CBL0137 sensitized GBM CSCs to radiotherapy using both in vitro and in vivo models. Treatment of CBL0137 combined with radiotherapy led to increased DNA damage in GBM patient specimens and failure to resolve the damage led to decreased cell viability. Using clonogenic assays, we confirmed that CBL0137 radiosensitized the CSCs. To validate that combination therapy impacted CSCs, we used an in vivo subcutaneous model and showed a decrease in the frequency of cancer stem cells present in tumors as well as decreased tumor volume. Using an orthotopic model of GBM, we confirmed that treatment with CBL0137 followed by radiotherapy led to significantly increased survival compared to either treatment alone. Radiotherapy remains a critical component of patient care for GBM, even though there exists a resistant subpopulation. Radio-sensitizing agents, including CBL0137, pose an exciting treatment paradigm to increase the efficacy of irradiation, especially by inclusively targeting CSCs.

scholarly journals A novel phosphoramide compound, DCZ0805, shows potent anti-myeloma activity via the NF-κB pathway

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Xuejie Gao ◽  
Bo Li ◽  
Anqi Ye ◽  
Houcai Wang ◽  
Yongsheng Xie ◽  
...  
Keyword(s):  
Mouse Model ◽  
Tumor Burden ◽  
High Rate ◽  
Cell Counting ◽  
Tumor Xenograft ◽  
Luciferase Reporter ◽  
Therapeutic Effects ◽  
Xenograft Mouse Model

Abstract Background Multiple myeloma (MM) is a highly aggressive and incurable clonal plasma cell disease with a high rate of recurrence. Thus, the development of new therapies is urgently needed. DCZ0805, a novel compound synthesized from osalmide and pterostilbene, has few observed side effects. In the current study, we intend to investigate the therapeutic effects of DCZ0805 in MM cells and elucidate the molecular mechanism underlying its anti-myeloma activity. Methods We used the Cell Counting Kit-8 assay, immunofluorescence staining, cell cycle assessment, apoptosis assay, western blot analysis, dual-luciferase reporter assay and a tumor xenograft mouse model to investigate the effect of DCZ0805 treatment both in vivo and in vitro. Results The results showed that DCZ0805 treatment arrested the cell at the G0/G1 phase and suppressed MM cells survival by inducing apoptosis via extrinsic and intrinsic pathways. DCZ0805 suppressed the NF-κB signaling pathway activation, which may have contributed to the inhibition of cell proliferation. DCZ0805 treatment remarkably reduced the tumor burden in the immunocompromised xenograft mouse model, with no obvious toxicity observed. Conclusion The findings of this study indicate that DCZ0805 can serve as a novel therapeutic agent for the treatment of MM.

scholarly journals Inhibition of Glycolysis Suppresses Cell Proliferation and Tumor Progression In Vivo: Perspectives for Chronotherapy

2021 ◽  
Vol 22 (9) ◽  
pp. 4390
Author(s):  
Jana Horváthová ◽  
Roman Moravčík ◽  
Miroslava Matúšková ◽  
Vladimír Šišovský ◽  
Andrej Boháč ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Tumor Progression ◽  
Umbilical Vein ◽  
Cell Metabolism ◽  
High Rate ◽  
Ki 67 ◽  
Immunodeficient Mice ◽  
Inhibition Of Glycolysis

A high rate of glycolysis is considered a hallmark of tumor progression and is caused by overexpression of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Therefore, we analyzed the possibility of inhibiting tumor and endothelial cell metabolism through the inhibition of PFKFB3 by a small molecule, (E)-1-(pyridin-4-yl)-3-(quinolin-2-yl)prop-2-en-1-one (PFK15), as a promising therapy. The effects of PFK15 on cell proliferation and apoptosis were analyzed on human umbilical vein endothelial cells (HUVEC) and the human colorectal adenocarcinoma cell line DLD1 through cytotoxicity and proliferation assays, flow cytometry, and western blotting. The results showed that PFK15 inhibited the proliferation of both cell types and induced apoptosis with decreasing the Bcl-2/Bax ratio. On the basis of the results obtained from in vitro experiments, we performed a study on immunodeficient mice implanted with DLD1 cells. We found a reduced tumor mass after morning PFK15 treatment but not after evening treatment, suggesting circadian control of underlying processes. The reduction in tumor size was related to decreased expression of Ki-67, a marker of cell proliferation. We conclude that inhibition of glycolysis can represent a promising therapeutic strategy for cancer treatment and its efficiency is circadian dependent.

scholarly journals Norepinephrine Protects against Methamphetamine Toxicity through β2-Adrenergic Receptors Promoting LC3 Compartmentalization

2021 ◽  
Vol 22 (13) ◽  
pp. 7232
Author(s):  
Gloria Lazzeri ◽  
Carla L. Busceti ◽  
Francesca Biagioni ◽  
Cinzia Fabrizi ◽  
Gabriele Morucci ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Light Chain ◽  
Adrenergic Receptors ◽  
Cell Damage ◽  
Protective Effects ◽  
Autophagy Flux ◽  
Brain Areas ◽  
Indirect Consequence

Norepinephrine (NE) neurons and extracellular NE exert some protective effects against a variety of insults, including methamphetamine (Meth)-induced cell damage. The intimate mechanism of protection remains difficult to be analyzed in vivo. In fact, this may occur directly on target neurons or as the indirect consequence of NE-induced alterations in the activity of trans-synaptic loops. Therefore, to elude neuronal networks, which may contribute to these effects in vivo, the present study investigates whether NE still protects when directly applied to Meth-treated PC12 cells. Meth was selected based on its detrimental effects along various specific brain areas. The study shows that NE directly protects in vitro against Meth-induced cell damage. The present study indicates that such an effect fully depends on the activation of plasma membrane β2-adrenergic receptors (ARs). Evidence indicates that β2-ARs activation restores autophagy, which is impaired by Meth administration. This occurs via restoration of the autophagy flux and, as assessed by ultrastructural morphometry, by preventing the dissipation of microtubule-associated protein 1 light chain 3 (LC3) from autophagy vacuoles to the cytosol, which is produced instead during Meth toxicity. These findings may have an impact in a variety of degenerative conditions characterized by NE deficiency along with autophagy impairment.

Sign in / Sign up

Export Citation Format

Share Document