Trafficking Defects of a Novel Autosomal Recessive Distal Renal Tubular Acidosis Mutant (S773P) of the Human Kidney Anion Exchanger (kAE1)
Bibliographic record
Abstract
Autosomal dominant and recessive distal renal tubular acidosis (dRTA) can be caused by mutations in the anion exchanger 1 (AE1 or SLC4A1) gene, which encodes the erythroid chloride/bicarbonate anion exchanger membrane glycoprotein (eAE1) and a truncated kidney isoform (kAE1). The biosynthesis and trafficking of kAE1 containing a novel recessive missense dRTA mutation (kAE1 S773P) was studied in transiently transfected HEK-293 cells, expressing the mutant alone or in combination with wild-type kAE1 or another recessive mutant, kAE1 G701D. The kAE1 S773P mutant was expressed at a three times lower level than wild-type, had a 2-fold decrease in its half-life, and was targeted for degradation by the proteasome. It could not be detected at the plasma membrane in human embryonic kidney cells and showed predominant endoplasmic reticulum immunolocalization in both human embryonic kidney and LLC-PK1 cells. The oligosaccharide on a kAE1 S773P N-glycosylation mutant (N555) was not processed to the complex form indicating impaired exit from the endoplasmic reticulum. The kAE1 S773P mutant showed decreased binding to an inhibitor affinity resin and increased sensitivity to proteases, suggesting that it was not properly folded. The other recessive dRTA mutant, kAE1 G701D, also exhibited defective trafficking to the plasma membrane. The recessive kAE1 mutants formed dimers like wild-type AE1 and could hetero-oligomerize with wild-type kAE1 or with each other. Hetero-oligomers of wild-type kAE1 with recessive kAE1 S773P or G701D, in contrast to the dominant kAE1 R589H mutant, were delivered to the plasma membrane. Autosomal dominant and recessive distal renal tubular acidosis (dRTA) can be caused by mutations in the anion exchanger 1 (AE1 or SLC4A1) gene, which encodes the erythroid chloride/bicarbonate anion exchanger membrane glycoprotein (eAE1) and a truncated kidney isoform (kAE1). The biosynthesis and trafficking of kAE1 containing a novel recessive missense dRTA mutation (kAE1 S773P) was studied in transiently transfected HEK-293 cells, expressing the mutant alone or in combination with wild-type kAE1 or another recessive mutant, kAE1 G701D. The kAE1 S773P mutant was expressed at a three times lower level than wild-type, had a 2-fold decrease in its half-life, and was targeted for degradation by the proteasome. It could not be detected at the plasma membrane in human embryonic kidney cells and showed predominant endoplasmic reticulum immunolocalization in both human embryonic kidney and LLC-PK1 cells. The oligosaccharide on a kAE1 S773P N-glycosylation mutant (N555) was not processed to the complex form indicating impaired exit from the endoplasmic reticulum. The kAE1 S773P mutant showed decreased binding to an inhibitor affinity resin and increased sensitivity to proteases, suggesting that it was not properly folded. The other recessive dRTA mutant, kAE1 G701D, also exhibited defective trafficking to the plasma membrane. The recessive kAE1 mutants formed dimers like wild-type AE1 and could hetero-oligomerize with wild-type kAE1 or with each other. Hetero-oligomers of wild-type kAE1 with recessive kAE1 S773P or G701D, in contrast to the dominant kAE1 R589H mutant, were delivered to the plasma membrane. The kidney plays an essential role in maintaining the acid-base balance of the body by reabsorbing bicarbonate in the proximal tubule and secreting acid into the urine. Impairment in acid secretion by α-intercalated cells of the distal nephron leads to the development of distal renal tubular acidosis (dRTA), 1The abbreviations used are: dRTA, distal renal tubular acidosis; AD dRTA, autosomal dominant distal renal tubular acidosis; AE, anion exchanger; AR dRTA, autosomal recessive dRTA; C12E8, octaethylene glycol mono-n-dodecyl ether; endo H, endoglycosidase H; GPA, glycophorin A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H2DIDS, 4,4′-diisothiocyanato-2,2′-dihydrostilbene disulfonate; HEK, human embryonic kidney; kAE1, kidney isoform of anion exchanger 1; kAE1 His, kAE1 with C-terminal His6 tag; LLC-PK1 cell, porcine kidney epithelial cells; MDCK, Madin-Darby canine kidney; PBS, phosphate-buffered saline; PFO, perfluoro-octanoic acid; PMSF, phenylmethylsulfonyl fluoride; SITS, 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonate; ER, endoplasmic reticulum; DMEM, Dulbecco's modified Eagle's medium; SAO, Southeast Asian ovalocytosis; HA, hemagglutinin; WT, wild type. 1The abbreviations used are: dRTA, distal renal tubular acidosis; AD dRTA, autosomal dominant distal renal tubular acidosis; AE, anion exchanger; AR dRTA, autosomal recessive dRTA; C12E8, octaethylene glycol mono-n-dodecyl ether; endo H, endoglycosidase H; GPA, glycophorin A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H2DIDS, 4,4′-diisothiocyanato-2,2′-dihydrostilbene disulfonate; HEK, human embryonic kidney; kAE1, kidney isoform of anion exchanger 1; kAE1 His, kAE1 with C-terminal His6 tag; LLC-PK1 cell, porcine kidney epithelial cells; MDCK, Madin-Darby canine kidney; PBS, phosphate-buffered saline; PFO, perfluoro-octanoic acid; PMSF, phenylmethylsulfonyl fluoride; SITS, 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonate; ER, endoplasmic reticulum; DMEM, Dulbecco's modified Eagle's medium; SAO, Southeast Asian ovalocytosis; HA, hemagglutinin; WT, wild type. which is inherited in both an autosomal dominant (AD dRTA) (1Karet F.E. Gainza F.J. Gyory A.Z. Unwin R.J. Wrong O. Tanner M.J.A. Nayir A. Alpay H. Santos F. Hulton S.A. Bakkaloglu A. Ozen S. Cunningham M.J. di Pietro A. Walker W.G. Lifton R.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6337-6342Crossref PubMed Scopus (230) Google Scholar, 2Bruce L.J. Cope D.L. Jones G.K. Schofield A.E. Burley M. Povey S. Unwin R.J. Wrong O. Tanner M.J. J. Clin. Invest. 1997; 100: 1693-1707Crossref PubMed Scopus (306) Google Scholar, 3Jarolim P. Shayakul C. Prabakaran D. Jiang L. Stuart-Tilley A. Rubin H.L. Simova S. Zavadil J. Herrin J.T. Brouillette J. Somers M.J. Seemanova E. Brugnara C. Guay-Woodford L.M. Alper S.L. J. Biol. Chem. 1998; 273: 6380-6388Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 4Bruce L.J. Wrong O. Toye A.M. Young M.T. Ogle G. Ismail Z. Sinha A.K. McMaster P. Hwaihwanje I. Nash G.B. Hart S. Lavu E. Palmer R. Othman A. Unwin R.J. Tanner M.J.A. Biochem. J. 2000; 350: 41-51Crossref PubMed Scopus (154) Google Scholar, 5Rysava R. Tesar V. Jirsa Jr., M. Brabec V. Jarolim P. Nephrol. Dial. Transplant. 1997; 12: 1869-1873Crossref PubMed Scopus (49) Google Scholar, 6Cheidde L. Vieira T.C. Lima P.R. Saad S.T. Heilberg I.P. Pediatrics. 2003; 112: 1361-1367Crossref PubMed Scopus (51) Google Scholar, 7Rungroj N. Devonald M.A. Cuthbert A.W. Reimann F. Akkarapatumwong V. Yenchitsomanus P.T. Bennett W.M. Karet F.E. J. Biol. Chem. 2004; 279: 13833-13838Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) and autosomal recessive manner (AR dRTA) (4Bruce L.J. Wrong O. Toye A.M. Young M.T. Ogle G. Ismail Z. Sinha A.K. McMaster P. Hwaihwanje I. Nash G.B. Hart S. Lavu E. Palmer R. Othman A. Unwin R.J. Tanner M.J.A. Biochem. J. 2000; 350: 41-51Crossref PubMed Scopus (154) Google Scholar, 8Tanphaichitr V.S. Sumboonnanonda A. Ideguchi H. Shayakul C. Brugnara C. Takao M. Veerakul G. Alper S.L. J. Clin. Invest. 1998; 102: 2173-2179Crossref PubMed Scopus (154) Google Scholar, 9Vasuvattakul S. Yenchitsomanus P.T. Vachuanichsanong P. Thuwajit P. Kaitwatcharachai C. Laosombat V. Malasit P. Wilairat P. Nimmannit S. Kidney Int. 1999; 56: 1674-1682Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 10Yenchitsomanus P.T. Vasuvattakul S. Kirdpon S. Wasanawatana S. Susaengrat W. Sreethiphayawan S. Chuawatana D. Mingkum S. Sawasdee N. Thuwajit P. Wilairat P. Malasit P. Nimmannit S. Am. J. Kidney Dis. 2002; 40: 21-29Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Patients with AD dRTA usually remain asymptomatic until adulthood, whereas AR dRTA patients are severe cases and early onset (11Karet F.E. Finberg K.E. Nelson R.D. Nayir A. Mocan H. Sanjad S.A. Rodriguez-Soriano J. Santos F. Cremers C.W. Di Pietro A. Hoffbrand B.I. Winiarski J. Bakkaloglu A. Ozen S. Dusunsel R. Goodyer P. Hulton S.A. Wu D.K. Skvorak A.B. Morton C.C. Cunningham M.J. Jha V. Lifton R.P. Nat. Genet. 1999; 21: 84-90Crossref PubMed Scopus (579) Google Scholar, 12Bajaj G. Quan A. Am. J. 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Seemanova E. Brugnara C. Guay-Woodford L.M. Alper S.L. J. Biol. Chem. 1998; 273: 6380-6388Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 4Bruce L.J. Wrong O. Toye A.M. Young M.T. Ogle G. Ismail Z. Sinha A.K. McMaster P. Hwaihwanje I. Nash G.B. Hart S. Lavu E. Palmer R. Othman A. Unwin R.J. Tanner M.J.A. Biochem. J. 2000; 350: 41-51Crossref PubMed Scopus (154) Google Scholar, 5Rysava R. Tesar V. Jirsa Jr., M. Brabec V. Jarolim P. Nephrol. Dial. Transplant. 1997; 12: 1869-1873Crossref PubMed Scopus (49) Google Scholar, 6Cheidde L. Vieira T.C. Lima P.R. Saad S.T. Heilberg I.P. Pediatrics. 2003; 112: 1361-1367Cr
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".