Advanced search options

Advanced Search Options 🞨

Browse by author name (“Author name starts with…”).

Find ETDs with:

in
/  
in
/  
in
/  
in

Written in Published in Earliest date Latest date

Sorted by

Results per page:

Sorted by: relevance · author · university · dateNew search

You searched for +publisher:"Case Western Reserve University" +contributor:("Gerken, Thomas"). Showing records 1 – 3 of 3 total matches.

Search Limiters

Last 2 Years | English Only

No search limiters apply to these results.

▼ Search Limiters

1. Revoredo, Leslie. Characterizing the (Glyco)peptide Substrate Specificities of the ppGalNAc T Family of Glycosyltransferases.

Degree: PhD, Chemistry, 2016, Case Western Reserve University

Many proteins of eukaryotic cells are known to be O-glycosylated. Glycoproteins with heavily O-glycosylated mucin domains provide important biological functions in a cell: i.e., protection from pathogens, cell-to-cell adhesion and intracellular protein trafficking. Mucin-type O-glycosylation occurs in the Golgi complex and begins with the transfer of GalNAc from UDP-GalNAc onto Ser/Thr residues of polypeptides. This step is catalyzed by a large family (20) called N-a-acetylgalactosaminyl transferases (ppGalNAc-T’s) and forms the GalNAc-a-O-Ser/Thr product. Subsequent elongation is performed by specific glycosyltransferases, producing a variety of glycans. Family members have been classified into peptide- and glycopeptide-preferring subfamilies, although both subfamilies possess variable activities against glycopeptide substrates. Structurally, 19 isoforms contain a C-terminal catalytic domain linked via a flexible linker to an N-terminal ricin-like lectin domain. The (glyco)peptide substrate specificities of the ppGalNAc-T transferases and the roles of the catalytic and lectin domains in glycopeptide glycosylation still remain largely unknown. Based on the systematic random peptide approach created by the Gerken Lab, I have determined the glycopeptide substrate specificities of several ppGalNAc-T isoforms. A series of (glyco)peptides were created in order to specifically probe the functions of the catalytic and lectin domains in terms of neighboring (1-5 residues) and remote prior glycosylation (6-17 residues) from an acceptor site, respectfully. Using several glycopeptide-preferring isoforms, glycosylation was observed from -4, -3, -1 and +1 relative to a neighboring GalNAc-O-Thr, which I attributed to specific GalNAc-O-Thr binding at the catalytic domain. The other series of glycopeptides contained a GalNAc-O-Thr near the C- or N- terminus of the substrate to address the directionality preferences of the lectin domain. Results with several peptide- and glycopeptide-preferring isoforms revealed preferences that varied among transferase isoform, where some preferred a C-terminally placed GalNAc-O-Thr, or a N-terminally placed GalNAc-O-Thr and others equally preferred the C-/N- terminally placed GalNAc-O-Thr. These directionality preferences are due to the GalNAc-O-Thr interactions at the lectin domain. Results of these studies revealed for the first time the site-specific glycopeptide glycosylation preferences by some ppGalNAc-T’s and has demonstrated that both domains of the ppGalNAc-T’s have specialized and unique functions that work in concert to control and order mucin-type O-glycosylation. Advisors/Committee Members: Gerken, Thomas (Advisor), Lee, Irene (Committee Chair).

Subjects/Keywords: Biochemistry; Chemistry; Mucin-type O-linked glycosylation; ppGalNAc T; lectin domain

Record DetailsSimilar RecordsGoogle PlusoneFacebookTwitterCiteULikeMendeleyreddit

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Revoredo, L. (2016). Characterizing the (Glyco)peptide Substrate Specificities of the ppGalNAc T Family of Glycosyltransferases. (Doctoral Dissertation). Case Western Reserve University. Retrieved from http://rave.ohiolink.edu/etdc/view?acc_num=case1464000374

Chicago Manual of Style (16th Edition):

Revoredo, Leslie. “Characterizing the (Glyco)peptide Substrate Specificities of the ppGalNAc T Family of Glycosyltransferases.” 2016. Doctoral Dissertation, Case Western Reserve University. Accessed February 26, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=case1464000374.

MLA Handbook (7th Edition):

Revoredo, Leslie. “Characterizing the (Glyco)peptide Substrate Specificities of the ppGalNAc T Family of Glycosyltransferases.” 2016. Web. 26 Feb 2020.

Vancouver:

Revoredo L. Characterizing the (Glyco)peptide Substrate Specificities of the ppGalNAc T Family of Glycosyltransferases. [Internet] [Doctoral dissertation]. Case Western Reserve University; 2016. [cited 2020 Feb 26]. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=case1464000374.

Council of Science Editors:

Revoredo L. Characterizing the (Glyco)peptide Substrate Specificities of the ppGalNAc T Family of Glycosyltransferases. [Doctoral Dissertation]. Case Western Reserve University; 2016. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=case1464000374


Case Western Reserve University

2. Perrine, Cynthia L. Profiling Glycosyltransferase Peptide Substrate Specificities: Studies on ppGalNAc T1, T2, T10, and T-synthase That Initiate Mucin-Type O-Glycosylation.

Degree: PhD, Chemistry, 2009, Case Western Reserve University

Many secreted and membrane-associated proteins of eukaryotic cells contain heavily O-glycosylated mucin domains. Glycoproteins which contain such domains play key biological functions, i.e., cell-cell adhesion, host pathogen interaction, and intracellular protein trafficking. Mucin type O-glycosylation occurs exclusively in the Golgi complex beginning with the transfer of GalNAc from UDP-GalNAc to a serine or threonine residue in the polypeptide catalyzed by a large family of polypeptide N-a-acetyl galactosaminyltransferases (ppGalNAcTs). The a-GalNAc-O-Ser/Thr product forms the core carbohydrate structure which is elongated in the Golgi by specific transferases. The peptide substrate specificities of these transferases are largely unknown. Based on earlier random peptide studies, I have determined the glycopeptide substrate specificities of the catalytic domains of ppGalNAc T1, T2, and T10 utilizing two complementary random glycopeptides, UDP-[3H]-GalNAc, and the azido-labeled UDP-GalNAc analogue, UDP-GalNAz. ppGalNAc T10 was shown to prefer Ser or Thr-O-GalNAc at the +1 position (relative to the site of glycosylation) with a large ~10 fold enhancement. ppGalNAc T1 and T2 gave little or no Ser/Thr-O-GalNAc preferences as expected from previous studies on these transferases. Peptide sequence and neighboring glycosylation effects on the specificity of T-synthase, which transfers ß Gal to the 3 position of GalNAc, was also determined using a random glycopeptide substrate. Novel enhancements for Phe and Tyr at the +3 and +4 positions relative to the site of glycosylation were observed with a smaller enhancement for Gly at the +1 position. An overall preference for negatively charged amino acids Glu and Asp was also observed. Neighboring glycosylation was also examined for T-synthase by glycosylating the porcine mucin tandem repeat. A candidate ppGalNAc T1 inhibitor peptide was also synthesized based on previous studies by our laboratory. The inhibitor peptide contained a photocross-linking p-nitro-phenylalanine derivative. The inhibitor peptide moderately decreased activity in the enzymes, but showed no specificity against ppGalNAc T1 or T2. As a result of these studies, robust methods for obtaining and quantifying the peptide and glycopeptide specificities of the individual transferases that initiate mucin O-glycosylation are now available. The development of methods for predicting transferase specific glycosylation are now possible based on this work. Advisors/Committee Members: Burda, Clemens (Committee Chair), Gerken, Thomas (Advisor).

Subjects/Keywords: Glycosylation; Glycoproteins; Mucins; UDP-GalNAc; ppGalNAcTs

Record DetailsSimilar RecordsGoogle PlusoneFacebookTwitterCiteULikeMendeleyreddit

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Perrine, C. L. (2009). Profiling Glycosyltransferase Peptide Substrate Specificities: Studies on ppGalNAc T1, T2, T10, and T-synthase That Initiate Mucin-Type O-Glycosylation. (Doctoral Dissertation). Case Western Reserve University. Retrieved from http://rave.ohiolink.edu/etdc/view?acc_num=case1253046997

Chicago Manual of Style (16th Edition):

Perrine, Cynthia L. “Profiling Glycosyltransferase Peptide Substrate Specificities: Studies on ppGalNAc T1, T2, T10, and T-synthase That Initiate Mucin-Type O-Glycosylation.” 2009. Doctoral Dissertation, Case Western Reserve University. Accessed February 26, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=case1253046997.

MLA Handbook (7th Edition):

Perrine, Cynthia L. “Profiling Glycosyltransferase Peptide Substrate Specificities: Studies on ppGalNAc T1, T2, T10, and T-synthase That Initiate Mucin-Type O-Glycosylation.” 2009. Web. 26 Feb 2020.

Vancouver:

Perrine CL. Profiling Glycosyltransferase Peptide Substrate Specificities: Studies on ppGalNAc T1, T2, T10, and T-synthase That Initiate Mucin-Type O-Glycosylation. [Internet] [Doctoral dissertation]. Case Western Reserve University; 2009. [cited 2020 Feb 26]. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=case1253046997.

Council of Science Editors:

Perrine CL. Profiling Glycosyltransferase Peptide Substrate Specificities: Studies on ppGalNAc T1, T2, T10, and T-synthase That Initiate Mucin-Type O-Glycosylation. [Doctoral Dissertation]. Case Western Reserve University; 2009. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=case1253046997


Case Western Reserve University

3. Miwa, Hazuki Eleanor. A Recombinant System to Model Proteoglycan Aggregate Interactions and Aggrecan Degradation.

Degree: PhD, Biochemistry, 2006, Case Western Reserve University

The proteoglycan aggregate is a major component of the articular cartilage extracellular matrix comprising hyaluronan (HA), aggrecan, and link protein. Aggrecan, heavily substituted with chondroitin sulfate (CS) and keratan sulfate (KS) glycosaminoglycans, contributes to cartilage hydration by binding water, thus lending articular cartilage its resistance to compressive deformation. A loss of aggrecan is observed in the early stages of osteoarthritis, which may relate to age-dependent changes in biochemical properties of proteoglycan aggregates. The research reported in this thesis was focused on producing recombinant molecules for modeling proteoglycan aggregate assembly and aggrecan proteolytic degradation. First, the proteoglycan tandem repeat (PTR) domains of link protein, which are responsible for HA binding, were expressed in E. coli to develop a tool for studying the PTR-HA interaction. Soluble monomeric PTR domains were obtained by a novel refolding procedure. Second, full-length recombinant link protein and aggrecan were expressed in a mammalian expression system as functional molecules capable of forming a ternary complex with HA. Furthermore, the expression of recombinant aggrecan in various mammalian cell lines allows the production of differently glycosylated aggrecans for comparison of glycosylation-specific functional differences. Last, both cartilage-derived and recombinant wild-type and mutagenized aggrecans were used to study proteinase-dependent aggrecan degradation observed in the cartilage extracellular matrix. The present work demonstrated that sulfated glycosaminoglycans covalently attached to both cartilage-derived and recombinant aggrecans regulate the susceptibility of aggrecan to ADAMTS4. A study using mutagenized recombinant aggrecan suggested that potentially glycosylated threonine and serine residues N-terminal and C-terminal to the ADAMTS4 cleavage site within the interglobular domain of aggrecan influence the rate of cleavage by ADAMTS4. The degree of hydrophobicity N-terminal to the ADAMTS4 cleavage site also affects aggrecan’s susceptibility to ADAMTS4. Recombinant aggrecan was substituted with KS by co-transfecting aggrecan with KS-sulfotransferases. KS-substituted aggrecan, which also had altered CS components, was found to be differentially susceptible to different isoforms of ADAMTS4. This work demonstrates the usefulness of the recombinant system for studies of molecular interactions with HA, aggrecan, and link protein, as well as for in vitro analyses of matrix proteinase-mediated degradation of aggrecan. Advisors/Committee Members: Gerken, Thomas (Advisor).

Subjects/Keywords: Chemistry, Biochemistry; Proteoglycan aggregates; Link protein; Aggrecan; ADAMTS4; MMP13; Aggrecanase

Record DetailsSimilar RecordsGoogle PlusoneFacebookTwitterCiteULikeMendeleyreddit

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Miwa, H. E. (2006). A Recombinant System to Model Proteoglycan Aggregate Interactions and Aggrecan Degradation. (Doctoral Dissertation). Case Western Reserve University. Retrieved from http://rave.ohiolink.edu/etdc/view?acc_num=case1130857836

Chicago Manual of Style (16th Edition):

Miwa, Hazuki Eleanor. “A Recombinant System to Model Proteoglycan Aggregate Interactions and Aggrecan Degradation.” 2006. Doctoral Dissertation, Case Western Reserve University. Accessed February 26, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=case1130857836.

MLA Handbook (7th Edition):

Miwa, Hazuki Eleanor. “A Recombinant System to Model Proteoglycan Aggregate Interactions and Aggrecan Degradation.” 2006. Web. 26 Feb 2020.

Vancouver:

Miwa HE. A Recombinant System to Model Proteoglycan Aggregate Interactions and Aggrecan Degradation. [Internet] [Doctoral dissertation]. Case Western Reserve University; 2006. [cited 2020 Feb 26]. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=case1130857836.

Council of Science Editors:

Miwa HE. A Recombinant System to Model Proteoglycan Aggregate Interactions and Aggrecan Degradation. [Doctoral Dissertation]. Case Western Reserve University; 2006. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=case1130857836

.