Eintrag weiter verarbeiten
Effect ofN-glycosylation on the transport activity of the peptide transporter PEPT1
Gespeichert in:
| Zeitschriftentitel: | American Journal of Physiology-Gastrointestinal and Liver Physiology |
|---|---|
| Personen und Körperschaften: | , , , , |
| In: | American Journal of Physiology-Gastrointestinal and Liver Physiology, 310, 2016, 2, S. G128-G141 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
American Physiological Society
|
| Schlagwörter: |
| author_facet |
Stelzl, Tamara Baranov, Tatjana Geillinger, Kerstin E. Kottra, Gabor Daniel, Hannelore Stelzl, Tamara Baranov, Tatjana Geillinger, Kerstin E. Kottra, Gabor Daniel, Hannelore |
|---|---|
| author |
Stelzl, Tamara Baranov, Tatjana Geillinger, Kerstin E. Kottra, Gabor Daniel, Hannelore |
| spellingShingle |
Stelzl, Tamara Baranov, Tatjana Geillinger, Kerstin E. Kottra, Gabor Daniel, Hannelore American Journal of Physiology-Gastrointestinal and Liver Physiology Effect ofN-glycosylation on the transport activity of the peptide transporter PEPT1 Physiology (medical) Gastroenterology Hepatology Physiology |
| author_sort |
stelzl, tamara |
| spelling |
Stelzl, Tamara Baranov, Tatjana Geillinger, Kerstin E. Kottra, Gabor Daniel, Hannelore 0193-1857 1522-1547 American Physiological Society Physiology (medical) Gastroenterology Hepatology Physiology http://dx.doi.org/10.1152/ajpgi.00350.2015 <jats:p>The intestinal peptide transporter PEPT1 provides bulk quantities of amino acids to epithelial cells. PEPT1 is a high-capacity and low-affinity solute carrier of the SLC15 family found in apical membranes of enterocytes in small intestine and distal colon. Surprisingly, murine PEPT1 (mPEPT1) has an apparent molecular mass of ∼95 kDa in the small intestine but ∼105 kDa in the large intestine. Here we describe studies on mPEPT1 protein glycosylation and how glycans affect transport function. Putative N-glycosylation sites of mPEPT1 were altered by site-directed mutagenesis followed by expression in Xenopus laevis oocytes. Replacement of six asparagine residues (N) at positions N50, N406, N439, N510, N515, and N532 by glutamine (Q) resulted in a decrease of the mPEPT1 mass by around 35 kDa. Electrophysiology revealed all glycosylation-deficient transporters to be functional with comparable expression levels in oocyte membranes. Strikingly, the mutant protein with N50Q exhibited a twofold decreased affinity for Gly-Sar but a 2.5-fold rise in the maximal inward currents compared with the wild-type protein. Elevated maximal transport currents were also recorded for cefadroxil and tri-l-alanine. Tracer flux studies performed with [<jats:sup>14</jats:sup>C]-Gly-Sar confirmed the reduction in substrate affinity and showed twofold increased maximal transport rates for the N50Q transporter. Elimination of individual N-glycosylation sites did not alter membrane expression in oocytes or overall transport characteristics except for the mutant protein N50Q. Because transporter surface density was not altered in N50Q, removal of the glycan at this location appears to accelerate the substrate turnover rate.</jats:p> Effect of<i>N</i>-glycosylation on the transport activity of the peptide transporter PEPT1 American Journal of Physiology-Gastrointestinal and Liver Physiology |
| doi_str_mv |
10.1152/ajpgi.00350.2015 |
| facet_avail |
Online Free |
| finc_class_facet |
Biologie Medizin |
| format |
ElectronicArticle |
| fullrecord |
blob:ai-49-aHR0cDovL2R4LmRvaS5vcmcvMTAuMTE1Mi9hanBnaS4wMDM1MC4yMDE1 |
| id |
ai-49-aHR0cDovL2R4LmRvaS5vcmcvMTAuMTE1Mi9hanBnaS4wMDM1MC4yMDE1 |
| institution |
DE-15 DE-Pl11 DE-Rs1 DE-105 DE-14 DE-Ch1 DE-L229 DE-D275 DE-Bn3 DE-Brt1 DE-Zwi2 DE-D161 DE-Gla1 DE-Zi4 |
| imprint |
American Physiological Society, 2016 |
| imprint_str_mv |
American Physiological Society, 2016 |
| issn |
0193-1857 1522-1547 |
| issn_str_mv |
0193-1857 1522-1547 |
| language |
English |
| mega_collection |
American Physiological Society (CrossRef) |
| match_str |
stelzl2016effectofnglycosylationonthetransportactivityofthepeptidetransporterpept1 |
| publishDateSort |
2016 |
| publisher |
American Physiological Society |
| recordtype |
ai |
| record_format |
ai |
| series |
American Journal of Physiology-Gastrointestinal and Liver Physiology |
| source_id |
49 |
| title |
Effect ofN-glycosylation on the transport activity of the peptide transporter PEPT1 |
| title_unstemmed |
Effect ofN-glycosylation on the transport activity of the peptide transporter PEPT1 |
| title_full |
Effect ofN-glycosylation on the transport activity of the peptide transporter PEPT1 |
| title_fullStr |
Effect ofN-glycosylation on the transport activity of the peptide transporter PEPT1 |
| title_full_unstemmed |
Effect ofN-glycosylation on the transport activity of the peptide transporter PEPT1 |
| title_short |
Effect ofN-glycosylation on the transport activity of the peptide transporter PEPT1 |
| title_sort |
effect of<i>n</i>-glycosylation on the transport activity of the peptide transporter pept1 |
| topic |
Physiology (medical) Gastroenterology Hepatology Physiology |
| url |
http://dx.doi.org/10.1152/ajpgi.00350.2015 |
| publishDate |
2016 |
| physical |
G128-G141 |
| description |
<jats:p>The intestinal peptide transporter PEPT1 provides bulk quantities of amino acids to epithelial cells. PEPT1 is a high-capacity and low-affinity solute carrier of the SLC15 family found in apical membranes of enterocytes in small intestine and distal colon. Surprisingly, murine PEPT1 (mPEPT1) has an apparent molecular mass of ∼95 kDa in the small intestine but ∼105 kDa in the large intestine. Here we describe studies on mPEPT1 protein glycosylation and how glycans affect transport function. Putative N-glycosylation sites of mPEPT1 were altered by site-directed mutagenesis followed by expression in Xenopus laevis oocytes. Replacement of six asparagine residues (N) at positions N50, N406, N439, N510, N515, and N532 by glutamine (Q) resulted in a decrease of the mPEPT1 mass by around 35 kDa. Electrophysiology revealed all glycosylation-deficient transporters to be functional with comparable expression levels in oocyte membranes. Strikingly, the mutant protein with N50Q exhibited a twofold decreased affinity for Gly-Sar but a 2.5-fold rise in the maximal inward currents compared with the wild-type protein. Elevated maximal transport currents were also recorded for cefadroxil and tri-l-alanine. Tracer flux studies performed with [<jats:sup>14</jats:sup>C]-Gly-Sar confirmed the reduction in substrate affinity and showed twofold increased maximal transport rates for the N50Q transporter. Elimination of individual N-glycosylation sites did not alter membrane expression in oocytes or overall transport characteristics except for the mutant protein N50Q. Because transporter surface density was not altered in N50Q, removal of the glycan at this location appears to accelerate the substrate turnover rate.</jats:p> |
| container_issue |
2 |
| container_start_page |
0 |
| container_title |
American Journal of Physiology-Gastrointestinal and Liver Physiology |
| container_volume |
310 |
| format_de105 |
Article, E-Article |
| format_de14 |
Article, E-Article |
| format_de15 |
Article, E-Article |
| format_de520 |
Article, E-Article |
| format_de540 |
Article, E-Article |
| format_dech1 |
Article, E-Article |
| format_ded117 |
Article, E-Article |
| format_degla1 |
E-Article |
| format_del152 |
Buch |
| format_del189 |
Article, E-Article |
| format_dezi4 |
Article |
| format_dezwi2 |
Article, E-Article |
| format_finc |
Article, E-Article |
| format_nrw |
Article, E-Article |
| _version_ |
1792339088370565124 |
| geogr_code |
not assigned |
| last_indexed |
2024-03-01T15:42:33.326Z |
| geogr_code_person |
not assigned |
| openURL |
url_ver=Z39.88-2004&ctx_ver=Z39.88-2004&ctx_enc=info%3Aofi%2Fenc%3AUTF-8&rfr_id=info%3Asid%2Fvufind.svn.sourceforge.net%3Agenerator&rft.title=Effect+ofN-glycosylation+on+the+transport+activity+of+the+peptide+transporter+PEPT1&rft.date=2016-01-15&genre=article&issn=1522-1547&volume=310&issue=2&pages=G128-G141&jtitle=American+Journal+of+Physiology-Gastrointestinal+and+Liver+Physiology&atitle=Effect+of%3Ci%3EN%3C%2Fi%3E-glycosylation+on+the+transport+activity+of+the+peptide+transporter+PEPT1&aulast=Daniel&aufirst=Hannelore&rft_id=info%3Adoi%2F10.1152%2Fajpgi.00350.2015&rft.language%5B0%5D=eng |
| SOLR | |
| _version_ | 1792339088370565124 |
| author | Stelzl, Tamara, Baranov, Tatjana, Geillinger, Kerstin E., Kottra, Gabor, Daniel, Hannelore |
| author_facet | Stelzl, Tamara, Baranov, Tatjana, Geillinger, Kerstin E., Kottra, Gabor, Daniel, Hannelore, Stelzl, Tamara, Baranov, Tatjana, Geillinger, Kerstin E., Kottra, Gabor, Daniel, Hannelore |
| author_sort | stelzl, tamara |
| container_issue | 2 |
| container_start_page | 0 |
| container_title | American Journal of Physiology-Gastrointestinal and Liver Physiology |
| container_volume | 310 |
| description | <jats:p>The intestinal peptide transporter PEPT1 provides bulk quantities of amino acids to epithelial cells. PEPT1 is a high-capacity and low-affinity solute carrier of the SLC15 family found in apical membranes of enterocytes in small intestine and distal colon. Surprisingly, murine PEPT1 (mPEPT1) has an apparent molecular mass of ∼95 kDa in the small intestine but ∼105 kDa in the large intestine. Here we describe studies on mPEPT1 protein glycosylation and how glycans affect transport function. Putative N-glycosylation sites of mPEPT1 were altered by site-directed mutagenesis followed by expression in Xenopus laevis oocytes. Replacement of six asparagine residues (N) at positions N50, N406, N439, N510, N515, and N532 by glutamine (Q) resulted in a decrease of the mPEPT1 mass by around 35 kDa. Electrophysiology revealed all glycosylation-deficient transporters to be functional with comparable expression levels in oocyte membranes. Strikingly, the mutant protein with N50Q exhibited a twofold decreased affinity for Gly-Sar but a 2.5-fold rise in the maximal inward currents compared with the wild-type protein. Elevated maximal transport currents were also recorded for cefadroxil and tri-l-alanine. Tracer flux studies performed with [<jats:sup>14</jats:sup>C]-Gly-Sar confirmed the reduction in substrate affinity and showed twofold increased maximal transport rates for the N50Q transporter. Elimination of individual N-glycosylation sites did not alter membrane expression in oocytes or overall transport characteristics except for the mutant protein N50Q. Because transporter surface density was not altered in N50Q, removal of the glycan at this location appears to accelerate the substrate turnover rate.</jats:p> |
| doi_str_mv | 10.1152/ajpgi.00350.2015 |
| facet_avail | Online, Free |
| finc_class_facet | Biologie, Medizin |
| format | ElectronicArticle |
| format_de105 | Article, E-Article |
| format_de14 | Article, E-Article |
| format_de15 | Article, E-Article |
| format_de520 | Article, E-Article |
| format_de540 | Article, E-Article |
| format_dech1 | Article, E-Article |
| format_ded117 | Article, E-Article |
| format_degla1 | E-Article |
| format_del152 | Buch |
| format_del189 | Article, E-Article |
| format_dezi4 | Article |
| format_dezwi2 | Article, E-Article |
| format_finc | Article, E-Article |
| format_nrw | Article, E-Article |
| geogr_code | not assigned |
| geogr_code_person | not assigned |
| id | ai-49-aHR0cDovL2R4LmRvaS5vcmcvMTAuMTE1Mi9hanBnaS4wMDM1MC4yMDE1 |
| imprint | American Physiological Society, 2016 |
| imprint_str_mv | American Physiological Society, 2016 |
| institution | DE-15, DE-Pl11, DE-Rs1, DE-105, DE-14, DE-Ch1, DE-L229, DE-D275, DE-Bn3, DE-Brt1, DE-Zwi2, DE-D161, DE-Gla1, DE-Zi4 |
| issn | 0193-1857, 1522-1547 |
| issn_str_mv | 0193-1857, 1522-1547 |
| language | English |
| last_indexed | 2024-03-01T15:42:33.326Z |
| match_str | stelzl2016effectofnglycosylationonthetransportactivityofthepeptidetransporterpept1 |
| mega_collection | American Physiological Society (CrossRef) |
| physical | G128-G141 |
| publishDate | 2016 |
| publishDateSort | 2016 |
| publisher | American Physiological Society |
| record_format | ai |
| recordtype | ai |
| series | American Journal of Physiology-Gastrointestinal and Liver Physiology |
| source_id | 49 |
| spelling | Stelzl, Tamara Baranov, Tatjana Geillinger, Kerstin E. Kottra, Gabor Daniel, Hannelore 0193-1857 1522-1547 American Physiological Society Physiology (medical) Gastroenterology Hepatology Physiology http://dx.doi.org/10.1152/ajpgi.00350.2015 <jats:p>The intestinal peptide transporter PEPT1 provides bulk quantities of amino acids to epithelial cells. PEPT1 is a high-capacity and low-affinity solute carrier of the SLC15 family found in apical membranes of enterocytes in small intestine and distal colon. Surprisingly, murine PEPT1 (mPEPT1) has an apparent molecular mass of ∼95 kDa in the small intestine but ∼105 kDa in the large intestine. Here we describe studies on mPEPT1 protein glycosylation and how glycans affect transport function. Putative N-glycosylation sites of mPEPT1 were altered by site-directed mutagenesis followed by expression in Xenopus laevis oocytes. Replacement of six asparagine residues (N) at positions N50, N406, N439, N510, N515, and N532 by glutamine (Q) resulted in a decrease of the mPEPT1 mass by around 35 kDa. Electrophysiology revealed all glycosylation-deficient transporters to be functional with comparable expression levels in oocyte membranes. Strikingly, the mutant protein with N50Q exhibited a twofold decreased affinity for Gly-Sar but a 2.5-fold rise in the maximal inward currents compared with the wild-type protein. Elevated maximal transport currents were also recorded for cefadroxil and tri-l-alanine. Tracer flux studies performed with [<jats:sup>14</jats:sup>C]-Gly-Sar confirmed the reduction in substrate affinity and showed twofold increased maximal transport rates for the N50Q transporter. Elimination of individual N-glycosylation sites did not alter membrane expression in oocytes or overall transport characteristics except for the mutant protein N50Q. Because transporter surface density was not altered in N50Q, removal of the glycan at this location appears to accelerate the substrate turnover rate.</jats:p> Effect of<i>N</i>-glycosylation on the transport activity of the peptide transporter PEPT1 American Journal of Physiology-Gastrointestinal and Liver Physiology |
| spellingShingle | Stelzl, Tamara, Baranov, Tatjana, Geillinger, Kerstin E., Kottra, Gabor, Daniel, Hannelore, American Journal of Physiology-Gastrointestinal and Liver Physiology, Effect ofN-glycosylation on the transport activity of the peptide transporter PEPT1, Physiology (medical), Gastroenterology, Hepatology, Physiology |
| title | Effect ofN-glycosylation on the transport activity of the peptide transporter PEPT1 |
| title_full | Effect ofN-glycosylation on the transport activity of the peptide transporter PEPT1 |
| title_fullStr | Effect ofN-glycosylation on the transport activity of the peptide transporter PEPT1 |
| title_full_unstemmed | Effect ofN-glycosylation on the transport activity of the peptide transporter PEPT1 |
| title_short | Effect ofN-glycosylation on the transport activity of the peptide transporter PEPT1 |
| title_sort | effect of<i>n</i>-glycosylation on the transport activity of the peptide transporter pept1 |
| title_unstemmed | Effect ofN-glycosylation on the transport activity of the peptide transporter PEPT1 |
| topic | Physiology (medical), Gastroenterology, Hepatology, Physiology |
| url | http://dx.doi.org/10.1152/ajpgi.00350.2015 |