lnu.sePublications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Design and engineering of human transferrin receptor 1 and its binding proteins
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. (Bjelic)ORCID iD: 0000-0001-7050-3445
2021 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The human transferrin receptor 1 (TfR) is central in maintaining adequate cellular iron levels with the iron carrier proteins transferrin (Tf) and ferritin (Ft). Known to bind TfR is also HFE, another iron regulatory protein, competing with Tf for receptor binding. Non-functional HFE leads to hereditary hemochromatosis, a disorder leading to unsustainable iron accumulation in most organs of the body. TfR is also expressed at the blood-brain barrier at high levels, therefore being one attractive target for development of non-invasive pharmaceutics delivery applications to the brain. Several diseases are caused by pathogens binding TfR. Among them malaria, caused by the parasite Plasmodium vivax, and several hemorrhagic fevers caused by zoonotic new world arenaviruses with rodent reservoirs in South America.

The TfR extracellular unit is structurally categorized in three domains, the helical, protease-like and apical domains. The helical domain is responsible for receptor dimerization. Transferrin is binding to the helical and protease-like domains, with an overlapping binding interface with HFE at the helical domain. The apical domain has structurally been shown to bind Ft, theattachment protein of P. vivax and Machupo virus glycoprotein 1 (MGP1).

This thesis focus is protein design and engineering of TfR and proteins interacting with TfR. The receptor domain where pathogens and Ft bind to TfR has been decoupled from the receptor and expressed in bacteria after utilizing computational protein design. The binding characteristics of MGP1 to TfR has been studied and optimized with directed evolution, showing a mutation that enhances binding affinity by 30-fold. Two small proteins have been designed with two different computational design strategies to bind TfR, at the pathogen and HFE binding sites. The two proteins, TB08 and TB14, are after directed evolution and mutational optimization binding to the TfR in a yeast surface display system.

Place, publisher, year, edition, pages
Växjö: Linnaeus University Press, 2021. , p. 52
Series
Linnaeus University Dissertations ; 406
Keywords [en]
Transferrin receptor, Machupo virus, protein engineering, Rosetta, flow cytometry, directed evolution
National Category
Biochemistry and Molecular Biology Theoretical Chemistry
Research subject
Natural Science, Biomedical Sciences
Identifiers
URN: urn:nbn:se:lnu:diva-100507Libris ID: n18bd2vflc84pf6pISBN: 978-91-89283-41-1 (electronic)ISBN: 978-91-89283-40-4 (print)OAI: oai:DiVA.org:lnu-100507DiVA, id: diva2:1521791
Public defence
2021-02-19, Lapiz (VI1158), Norra kajplanen 6, Kalmar, 10:00 (English)
Opponent
Supervisors
Available from: 2021-01-25 Created: 2021-01-25 Last updated: 2024-03-06Bibliographically approved
List of papers
1. Computational backbone design enables soluble engineering of transferrin receptor apical domain
Open this publication in new window or tab >>Computational backbone design enables soluble engineering of transferrin receptor apical domain
2020 (English)In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 88, no 12, p. 1569-1577Article in journal (Refereed) Published
Abstract [en]

Supply of iron into human cells is achieved by iron carrier protein transferrin and its receptor that upon complex formation get internalized by endocytosis. Similarly, the iron needs to be delivered into the brain, and necessitates the transport across the blood-brain barrier. While there are still unanswered questions about these mechanisms, extensive efforts have been made to use the system for delivery of therapeutics into biological compartments. The dimeric form of the receptor, where each subunit consists of three domains, further complicates the detailed investigation of molecular determinants responsible for guiding the receptor interactions with other proteins. Especially the apical domain's biological function has been elusive. To further the study of transferrin receptor, we have computationally decoupled the apical domain for soluble expression, and validated the design strategy by structure determination. Besides presenting a methodology for solubilizing domains, the results will allow for study of apical domain's function.

Place, publisher, year, edition, pages
John Wiley & Sons, 2020
Keywords
iron transport, protein design, Rosetta, transferrin, transferrin receptor
National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-97273 (URN)10.1002/prot.25974 (DOI)000548420100001 ()32592192 (PubMedID)2-s2.0-85087825287 (Scopus ID)
Available from: 2020-07-23 Created: 2020-07-23 Last updated: 2023-05-31Bibliographically approved
2. Tuning the binding interface between Machupo virus glycoprotein and human transferrin receptor
Open this publication in new window or tab >>Tuning the binding interface between Machupo virus glycoprotein and human transferrin receptor
2021 (English)In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 89, no 3, p. 311-321Article in journal (Refereed) Published
Abstract [en]

Machupo virus, known to cause hemorrhagic fevers, enters human cells via binding with its envelope glycoprotein to transferrin receptor 1 (TfR). Similarly, the receptor interactions have been explored in biotechnological applications as a molecular system to ferry therapeutics across the cellular membranes and through the impenetrable blood-brain barrier that effectively blocks any such delivery into the brain. Study of the experimental structure of Machupo virus glycoprotein 1 (MGP1) in complex with TfR and glycoprotein sequence homology has identified some residues at the interface that influence binding. There are, however, no studies that have attempted to optimize the binding potential between MGP1 and TfR. In pursuits for finding therapeutic solutions for the New World arenaviruses, and to gain a greater understanding of MGP1 interactions with TfR, it is crucial to understand the structure-sequence relationship driving the interface formation. By displaying MGP1 on yeast surface we have examined the contributions of individual residues to the binding of solubilized ectodomain of TfR. We identified MGP1 binding hot spot residues, assessed the importance of posttranslational N-glycan modifications, and used a selection with random mutagenesis for affinity maturation. We show that the optimized MGP1 variants can bind more strongly to TfR than the native MGP1, and there is an MGP1 sequence that retains binding in the absence of glycosylation, but with the addition of further amino acid substitutions. The engineered variants can be used to probe cellular internalization or the blood-brain barrier crossing to achieve greater understanding of TfR mediated internalization.

Place, publisher, year, edition, pages
John Wiley & Sons, 2021
Keywords
blood-brain barrier, flow cytometry, fluorescence-activated cell sorting, Machupo virus glycoprotein 1, Rosetta, transferrin receptor, yeast surface display
National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:lnu:diva-99618 (URN)10.1002/prot.26016 (DOI)000583774500001 ()33068039 (PubMedID)2-s2.0-85093666536 (Scopus ID)2020 (Local ID)2020 (Archive number)2020 (OAI)
Available from: 2021-01-12 Created: 2021-01-12 Last updated: 2023-05-31Bibliographically approved

Open Access in DiVA

Doctoral Dissertation (Comprehensive Summary)(21558 kB)1232 downloads
File information
File name FULLTEXT01.pdfFile size 21558 kBChecksum SHA-512
238ae87b77794dcf554b5166c6cb68def0b9c66eabad3b6046b7558d4be27c69ff7080186c2a0739e4ecc401b5db16841bb8ad6b6598b25373d104f24820a381
Type fulltextMimetype application/pdf

Other links

Buy Book (SEK 250 + VAT and postage) lnupress@lnu.se

Authority records

Sjöström, Dick J.

Search in DiVA

By author/editor
Sjöström, Dick J.
By organisation
Department of Chemistry and Biomedical Sciences
Biochemistry and Molecular BiologyTheoretical Chemistry

Search outside of DiVA

GoogleGoogle Scholar
Total: 1234 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 1110 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf