Dissecting OGT's TPR domain to identify determinants of cellular function.

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that glycosylates myriad intracellular proteins and cleaves the transcriptional coregulator Host Cell Factor 1 to regulate cell cycle processes. Via these catalytic activities as well as noncatalytic protein-protein interactions, OGT maintains cell homeostasis. OGT's tetratricopeptide repeat (TPR) domain is important in substrate recognition, but there is little information on how changing the TPR domain impacts its cellular functions. Here, we investigate how altering OGT's TPR domain impacts cell growth after the endogenous enzyme is deleted. We find that disrupting the TPR residues required for OGT dimerization leads to faster cell growth, whereas truncating the TPR domain slows cell growth. We also find that OGT requires eight of its 13 TPRs to sustain cell viability. OGT-8, like the nonviable shorter OGT variants, is mislocalized and has reduced Ser/Thr glycosylation activity; moreover, its interactions with most of wild-type OGT's binding partners are broadly attenuated. Therefore, although OGT's five N-terminal TPRs are not essential for cell viability, they are required for proper subcellular localization and for mediating many of OGT's protein-protein interactions. Because the viable OGT truncation variant we have identified preserves OGT's essential functions, it may facilitate their identification.

Phospho-KNL-1 recognition by a TPR domain targets the BUB-1-BUB-3 complex to C. elegans kinetochores.

During mitosis, the Bub1-Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide repeat (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1-Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated "MELT" motifs in the kinetochore scaffold protein Knl1. Motivated by the greater phenotypic severity of BUB-1 versus BUB-3 loss in C. elegans, we show that the BUB-1 TPR domain directly recognizes a distinct class of phosphorylated motifs in KNL-1 and that this interaction is essential for BUB-1-BUB-3 localization and function. BUB-3 recognition of phospho-MELT motifs additively contributes to drive super-stoichiometric accumulation of BUB-1-BUB-3 on its KNL-1 scaffold during mitotic entry. Bub1's TPR domain interacts with Knl1 in other species, suggesting that collaboration of TPR-dependent and Bub3-dependent interfaces in Bub1-Bub3 localization and functions may be conserved.

MdTPR16, an apple tetratricopeptide repeat (TPR)-like superfamily gene, positively regulates drought stress in apple.

The Tetratricopeptide repeat (TPR)-like superfamily with TPR conserved domains is widely involved in the growth and abiotic stress in many plants. In this report, the gene MdTPR16 belongs to the TPR family in apple (Malus domestica). Promoter analysis reveal that MdTPR16 incorporated various stress response elements, including the drought stress response elements. And different abiotic stress treatments, drought especially, significantly induce the response of MdTPR16. Overexpression of MdTPR16 result in better drought tolerance in apple and Arabidopsis by up-regulating the expression levels of drought stress-related genes, achieving a higher chlorophyll content level, more material accumulation, and overall better growth compared to WT in the drought. Under drought stress, the overexpressed MdTPR16 also mitigate the oxidative damage in cells by reducing the electrolyte leakage, malondialdehyde content, and the H2O2 and O2- accumulation in apples and Arabidopsis. In conclusion, MdTPR16 act as a beneficial regulator of drought stress response by regulating the expression of related genes and the cumulation of reactive oxygen species (ROS).

Interferon-induced protein with tetratricopeptide repeats 5 of black fruit bat (Pteropus alecto) displays a broad inhibition of RNA viruses.

Bats are natural host reservoirs and have adapted a unique innate immune system that permits them to host many viruses without exhibiting symptoms. Notably, bat interferon stimulated genes (ISGs) have been shown to play antiviral roles. Interferon induced protein with tetratricopeptide repeats 5 (IFIT5) is a well-characterised ISG in humans with antiviral activities against negative-sense RNA viruses via inhibiting viral transcription. Here, we aim to investigate if Pteropus alecto (pa) IFIT5 (paIFIT5) possess the ability to inhibit negative-sense RNA viruses. Initially, gene syntenic and comparative structural analyses of multiple animals highlighted a high level of similarity between Pteropus alecto and human IFIT5 proteins. Our results showed that paIFIT5 was significantly inducible by viral and dsRNA stimulation. Transient overexpression of paIFIT5 inhibited the replication of vesicular stomatitis virus (VSV). Using minireplicon and transcription reporter assays, we demonstrated the ability of paIFIT5 specifically to inhibit H17N10 polymerase activity. Mechanistically, we noticed that the antiviral potential of paIFIT5 against negative sense RNA viruses was retributed to its interaction with 5'ppp containing RNA. Taken together, these findings highlight the genetic and functional conservation of IFIT5 among mammals.

Tanc1/2 TPR domain interacts with Myo18a C-terminus and undergoes liquid-liquid phase separation.

Tanc1 and its homologous protein Tanc2 are critical synaptic scaffold proteins which regulate synaptic spine densities and excitatory synapse strength. Recent studies indicated TANC1 and TANC2 are candidate genes of several neurodevelopmental disorders (NDDs). In this study, we identified and characterized a novel interaction between Tanc1/2 and Myo18a, mediated by the Tanc1/2 TPR domains and Myo18a coiled-coil domain and C-extension (CCex). Sequence analysis and size exclusion chromatography experiments reveal that high salt disrupts the interaction between Myo18a and Tanc1/2, indicating that the interaction is primarily driven by charge-charge interactions. More importantly, we found that the Tanc1-TPR/Myo18a CCex interaction could undergo liquid-liquid phase separation (LLPS) in both cultured cells and test tubes, which provides the biochemical basis and potential molecular mechanisms for the LLPS-mediated interactions between Myo18a and Tanc1/2.

ZmTDM1 encodes a tetratricopeptide repeat domain protein and is required for meiotic exit in maize.

Elaborate cell-cycle control must be adopted to ensure the continuity of the meiotic second division and termination after that. Despite its importance, however, the genetic controls underlying the meiotic cell cycle have not been reported in maize. Here, we characterized a meiotic cell-cycle controller ZmTDM1, which is a homolog of Arabidopsis TDM1 and encodes a canonical tetratricopeptide repeat domain protein in maize. The Zmtdm1 homozygous plants exhibited complete male sterility and severe female abortion. In Zmtdm1 mutants, cell-cycle progression was almost identical to that of wild type from leptotene to anaphase II. However, chromosomes in the tetrad failed meiotic termination at the end of the second division and underwent additional divisions in succession without DNA replication, reducing the ploidy to less than haploid in the product. In addition, two ZmTDM1-like homologs (ZmTDML1 and ZmTDML2) were not functional in meiotic cell-cycle control. Moreover, ZmTDM1 interacted with RING-type E3 ubiquitin ligase, revealing that it acts as a subunit of the APC/C E3 ubiquitin ligase complex. Overall, our results identified a regulator of meiotic cell cycle in maize and demonstrated that ZmTDM1 is essential for meiotic exit after meiosis II.

TTC7B is a new prognostic biomarker in head and neck squamous cell carcinoma linked to immune infiltration and ferroptosis.

OBJECTIVE: To investigate the expression of TTC7B and its prognostic significance, biological roles, and impact on the immune system in patients with head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: Clinical and genomic data were obtained from TCGA (The Cancer Genome Atlas), GEO (Gene Expression Omnibus), GEPIA2 (Gene Expression Profiling Interactive Analysis 2.0), and TIMER2.0 (Tumor Immune Estimation Resource 2.0) databases. R software was utilized to process the retrieved data. qPCR and immunohistochemical assays were performed to validate the findings obtained from the databases. RESULTS: High expression of TTC7B was observed in HNSCC, and this heightened expression is significantly associated with reduced overall survival (OS) in patients, making it an independent risk factor impacting OS. TTC7B is correlated with focal adhesions and cell migration pathways based on functional enrichment analysis. CIBERSORT analysis and TIMER2.0 show a positive link between TTC7B and multiple immune cells, particularly macrophages. Pearson's analysis reveals a significant correlation between TTC7B and ferroptosis-related genes. CONCLUSION: In all, TTC7B could serve as a promising prognostic indicator of HNSCC, and is closely associated with focal adhesions, immune infiltration, and ferroptosis.

Roles of interferon induced protein with tetratricopeptide repeats (IFIT) family in autoimmune disease.

Interferon-induced tetrapeptide repeat (IFIT) family proteins are an important component of the antiviral immune response. There are four known members of the human IFIT family, namely IFIT1, IFIT2, IFIT3 and IFIT5. More and more evidence shows that IFIT family members are involved in a variety of pathophysiological processes in vivo, regulate the homeostasis and differentiation of a variety of cells including immune cells, and are closely related to a variety of autoimmune diseases, which is expected to become a new therapeutic target. This review reviews the biological roles of different IFIT proteins in various autoimmune diseases, and highlights the potential use of these molecules as biomarkers and prognostic factors in autoimmune diseases, with a view to providing ideas for exploring the diagnosis and treatment of autoimmune diseases.

The Interferon-Induced Protein with Tetratricopeptide Repeats Repress Influenza Virus Infection by Inhibiting Viral RNA Synthesis.

Influenza A virus (IAV) is an eight-segment negative-sense RNA virus and is subjected to gene recombination between strains to form novel strains, which may lead to influenza pandemics. Seasonal influenza occurs annually and causes great losses in public healthcare. In this study, we examined the role of interferon-induced protein with tetratricopeptide repeats 1 and 2 (IFIT1 and IFIT2) in influenza virus infection. Knockdown of IFIT1 or IFIT2 using a lentiviral shRNA increased viral nucleoprotein (NP) and nonstructural protein 1 (NS1) protein levels, as well as progeny virus production in A/Puerto Rico/8/34 H1N1 (PR/8)-infected lung epithelial A549 cells. Overexpression of IFIT1 or IFIT2 reduced viral NP and NS1 RNA and protein levels in PR/8-infected HEK293 cells. Overexpression of IFIT1 or IFIT2 also inhibited influenza virus infection of various H1N1 strains, including PR/8, A/WSN/1933, A/California/07/2009 and A/Oklahoma/3052/2009, as determined by a viral reporter luciferase assay. Furthermore, knockdown of IFIT1 or IFIT2 increased while overexpression of IFIT1 or IFIT2 decreased viral RNA, complementary RNA, and mRNA levels of NP and NS1, as well as viral polymerase activities. Taken together, our results support that both IFIT1 and -2 have anti-influenza virus activities by inhibiting viral RNA synthesis.

A Biallelic Truncating Variant in the TPR Domain of GEMIN5 Associated with Intellectual Disability and Cerebral Atrophy.

GEMIN5 is a multifunctional RNA-binding protein required for the assembly of survival motor neurons. Several bi-allelic truncating and missense variants in this gene are reported to cause a neurodevelopmental disorder characterized by cerebellar atrophy, intellectual disability (ID), and motor dysfunction. Whole exome sequencing of a Pakistani consanguineous family with three brothers affected by ID, cerebral atrophy, mobility, and speech impairment revealed a novel homozygous 3bp-deletion NM_015465.5:c.3162_3164del that leads to the loss of NM_015465.5 (NP_056280.2):p. (Asp1054_Ala1055delinsGlu) amino acid in one of the α-helixes of the tetratricopeptide repeats of GEMIN5. In silico 3D representations of the GEMIN5 dimerization domain show that this variant likely affects the orientation of the downstream sidechains out of the helix axis, which would affect the packing with neighboring helices. The phenotype of all affected siblings overlaps well with previously reported patients, suggesting that NM_015465.5: c.3162_3164del (NP_056280.2):p. (Asp1054_Ala1055delinsGlu) is a novel GEMIN5 pathogenic variant. Overall, our data expands the molecular and clinical phenotype of the recently described neurodevelopmental disorder with cerebellar atrophy and motor dysfunction (NEDCAM) syndrome.

Tetratricopeptide repeat protein SlREC2 positively regulates cold tolerance in tomato.

Cold stress is a key environmental constraint that dramatically affects the growth, productivity, and quality of tomato (Solanum lycopersicum); however, the underlying molecular mechanisms of cold tolerance remain poorly understood. In this study, we identified REDUCED CHLOROPLAST COVERAGE 2 (SlREC2) encoding a tetratricopeptide repeat protein that positively regulates tomato cold tolerance. Disruption of SlREC2 largely reduced abscisic acid (ABA) levels, photoprotection, and the expression of C-REPEAT BINDING FACTOR (CBF)-pathway genes in tomato plants under cold stress. ABA deficiency in the notabilis (not) mutant, which carries a mutation in 9-CIS-EPOXYCAROTENOID DIOXYGENASE 1 (SlNCED1), strongly inhibited the cold tolerance of SlREC2-silenced plants and empty vector control plants and resulted in a similar phenotype. In addition, foliar application of ABA rescued the cold tolerance of SlREC2-silenced plants, which confirms that SlNCED1-mediated ABA accumulation is required for SlREC2-regulated cold tolerance. Strikingly, SlREC2 physically interacted with ß-RING CAROTENE HYDROXYLASE 1b (SlBCH1b), a key regulatory enzyme in the xanthophyll cycle. Disruption of SlBCH1b severely impaired photoprotection, ABA accumulation, and CBF-pathway gene expression in tomato plants under cold stress. Taken together, this study reveals that SlREC2 interacts with SlBCH1b to enhance cold tolerance in tomato via integration of SlNCED1-mediated ABA accumulation, photoprotection, and the CBF-pathway, thus providing further genetic knowledge for breeding cold-resistant tomato varieties.

How Different Pathologies Are Affected by IFIT Expression.

The type-I interferon (IFN) system represents the first line of defense against viral pathogens. Recognition of the virus initiates complex signaling pathways that result in the transcriptional induction of IFNs, which are then secreted. Secreted IFNs stimulate nearby cells and result in the production of numerous proinflammatory cytokines and antiviral factors. Of particular note, IFN-induced tetratricopeptide repeat (IFIT) proteins have been thoroughly studied because of their antiviral activity against different viral pathogens. Although classically studied as an antiviral protein, IFIT expression has recently been investigated in the context of nonviral pathologies, such as cancer and sepsis. In oral squamous cell carcinoma (OSCC), IFIT1 and IFIT3 promote metastasis, while IFIT2 exhibits the opposite effect. The role of IFIT proteins during bacterial/fungal sepsis is still under investigation, with studies showing conflicting roles for IFIT2 in disease severity. In the setting of viral sepsis, IFIT proteins play a key role in clearing viral infection. As a result, many viral pathogens, such as SARS-CoV-2, employ mechanisms to inhibit the type-I IFN system and promote viral replication. In cancers that are characterized by upregulated IFIT proteins, medications that decrease IFIT expression may reduce metastasis and improve survival rates. Likewise, in cases of viral sepsis, therapeutics that increase IFIT expression may improve viral clearance and reduce the risk of septic shock. By understanding the effect of IFIT proteins in different pathologies, novel therapeutics can be developed to halt disease progression.

Differential expression of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) in Alzheimer's disease and HIV-1 associated neurocognitive disorders.

The United Nations projects that one in every six people will be over the age of 65 by the year 2050. With a rapidly aging population, the risk of Alzheimer's disease (AD) becomes a major concern. AD is a multifactorial disease that involves neurodegeneration in the brain with mild dementia and deficits in memory and other cognitive domains. Additionally, it has been established that individuals with Human Immunodeficiency Virus-1 (HIV-1) experience a 5 to 10-year accelerated aging and an increased risk of developing HIV-associated neurocognitive disorders (HAND). Despite a significant amount of clinical evidence pointing towards a potential overlap between neuropathogenic processes in HAND and AD, the underlying epigenetic link between these two diseases is mostly unknown. This study is focused on identifying differentially expressed genes observed in both AD and HAND using linear regression models and a more robust significance analysis of microarray. The results established that the dysregulated type 1 and 2 interferon pathways observed in both AD and HAND contribute to the similar pathologies of these diseases within the brain. The current study identifies the important roles of interferon pathways in AD and HAND, a relationship that may be useful for earlier detection in the future.

IFN-Induced Protein with Tetratricopeptide Repeats 2 Limits Autoimmune Inflammation by Regulating Myeloid Cell Activation and Metabolic Activity.

Besides antiviral functions, type I IFN expresses potent anti-inflammatory properties and is being widely used to treat certain autoimmune conditions, such as multiple sclerosis. In a murine model of multiple sclerosis, experimental autoimmune encephalomyelitis, administration of IFN-ß effectively attenuates the disease development. However, the precise mechanisms underlying IFN-ß-mediated treatment remain elusive. In this study, we report that IFN-induced protein with tetratricopeptide repeats 2 (Ifit2), a type I and type III IFN-stimulated gene, plays a previously unrecognized immune-regulatory role during autoimmune neuroinflammation. Mice deficient in Ifit2 displayed greater susceptibility to experimental autoimmune encephalomyelitis and escalated immune cell infiltration in the CNS. Ifit2 deficiency was also associated with microglial activation and increased myeloid cell infiltration. We also observed that myelin debris clearance and the subsequent remyelination were substantially impaired in Ifit2-/- CNS tissues. Clearing myelin debris is an important function of the reparative-type myeloid cell subset to promote remyelination. Indeed, we observed that bone marrow-derived macrophages, CNS-infiltrating myeloid cells, and microglia from Ifit2-/- mice express cytokine and metabolic genes associated with proinflammatory-type myeloid cell subsets. Taken together, our findings uncover a novel regulatory function of Ifit2 in autoimmune inflammation in part by modulating myeloid cell function and metabolic activity.

Structural insights into the regulation of Cas7-11 by TPR-CHAT.

The CRISPR-guided caspase (Craspase) complex is an assembly of the target-specific RNA nuclease known as Cas7-11 bound to CRISPR RNA (crRNA) and an ancillary protein known as TPR-CHAT (tetratricopeptide repeats (TPR) fused with a CHAT domain). The Craspase complex holds promise as a tool for gene therapy and biomedical research, but its regulation is poorly understood. TPR-CHAT regulates Cas7-11 nuclease activity via an unknown mechanism. In the present study, we use cryoelectron microscopy to determine structures of the Desulfonema magnum (Dm) Craspase complex to gain mechanistic insights into its regulation. We show that DmTPR-CHAT stabilizes crRNA-bound DmCas7-11 in a closed conformation via a network of interactions mediated by the DmTPR-CHAT N-terminal domain, the DmCas7-11 insertion finger and Cas11-like domain, resulting in reduced target RNA accessibility and cleavage.

Bioinformatics identification of miR-514b-5p promotes NSCLC progression and induces PI3K/AKT and p38 pathways by targeting small glutamine-rich tetratricopeptide repeat-containing protein beta.

Lung cancer is the most aggressive cancer with the highest mortality and incidence rates worldwide. MicroRNAs have been identified as potential targets for non-small cell lung cancer (NSCLC) treatment. However, the modulatory role of miR-514b-5p in NSCLC progression is little known. In the present study, miRNA expression datasets for NSCLC were downloaded from the Cancer Genome Atlas and Gene Ontology Omnibus databases. Gene expression was assessed using a quantitative real-time PCR, and western blot analysis and immunohistochemical staining was used to determine protein expression. Gain and loss of function experiments were performed to investigate the impact of miR-514b-5p and small glutamine-rich tetratricopeptide repeat-containing protein beta (SGTB) on cell proliferation and apoptosis. RNA immunoprecipitation and dual-luciferase assays were performed to analyse the target gene of miR-514b-5p. The biological roles of miR-514b-5p were lastly evaluated using nude mouse tumorigenicity assays in vivo. We found that miR-514b-5p was dramatically increased in NSCLC tissues and higher miR-514b-5p expression was associated with poorer overall survival in NSCLC patients. Furthermore, overexpression of miR-514b-5p promoted NSCLC cell growth and suppressed apoptosis by inducing the activation of the phosphatidylinositol-3-kinase (PI3K)/AKT and p38 signalling pathways. Mechanistically, dual-luciferase and the RNA immunoprecipitation results highlighted that SGTB was a target gene of miR-514b-5p. Moreover, overexpression of SGTB reduced cell division and promoted apoptosis in vitro through blocking the PI3K/AKT and p38 signalling pathways. Our findings indicated that miR-514b-5p contributes to carcinoma progression in NSCLC via the PI3K/AKT and p38 signalling pathways by targeting SGTB and this could be a promising diagnostic and therapeutic target for the treatment of NSCLC.

The TPR domain of PgaA is a multifunctional scaffold that binds PNAG and modulates PgaB-dependent polymer processing.

The synthesis of exopolysaccharides as biofilm matrix components by pathogens is a crucial factor for chronic infections and antibiotic resistance. Many periplasmic proteins involved in polymer processing and secretion in Gram-negative synthase dependent exopolysaccharide biosynthetic systems have been individually characterized. The operons responsible for the production of PNAG, alginate, cellulose and the Pel polysaccharide each contain a gene that encodes an outer membrane associated tetratricopeptide repeat (TPR) domain containing protein. While the TPR domain has been shown to bind other periplasmic proteins, the functional consequences of these interactions for the polymer remain poorly understood. Herein, we show that the C-terminal TPR region of PgaA interacts with the de-N-acetylase domain of PgaB, and increases its deacetylase activity. Additionally, we found that when the two proteins form a complex, the glycoside hydrolase activity of PgaB is also increased. To better understand structure-function relationships we determined the crystal structure of a stable TPR module, which has a conserved groove formed by three repeat motifs. Tryptophan quenching, mass spectrometry analysis and molecular dynamics simulation studies suggest that the crystallized TPR module can bind PNAG/dPNAG via its electronegative groove on the concave surface, and potentially guide the polymer through the periplasm towards the porin for export. Our results suggest a scaffolding role for the TPR domain that combines PNAG/dPNAG translocation with the modulation of its chemical structure by PgaB.

The tetratricopeptide repeat protein OsTPR075 promotes heading by regulating florigen transport in rice.

Rice (Oryza sativa) is one of the most important crops worldwide. Heading date is a vital agronomic trait that influences rice yield and adaption to local conditions. Hd3a, a proposed florigen that primarily functions under short-day (SD) conditions, is a mobile flowering signal that promotes the floral transition in rice. Nonetheless, how Hd3a is transported from leaves to the shoot apical meristem (SAM) under SDs remains elusive. Here, we report that FT-INTERACTING PROTEIN9 (OsFTIP9) specifically regulates rice flowering time under SDs by facilitating Hd3a transport from companion cells (CCs) to sieve elements (SEs). Furthermore, we show that the tetratricopeptide repeat (TPR) protein OsTPR075 interacts with both OsFTIP9 and OsFTIP1 and strengthens their respective interactions with Hd3a and the florigen RICE FLOWERING LOCUS T1 (RFT1). This in turn affects the trafficking of Hd3a and RFT1 to the SAM, thus regulating flowering time under SDs and long-day conditions, respectively. Our findings suggest that florigen transport in rice is mediated by different OsFTIPs under different photoperiods and those interactions between OsTPR075 and OsFTIPs are essential for mediating florigen movement from leaves to the SAM.

Tetratricopeptide repeat domain 36 protects renal tubular cells from cisplatin-induced apoptosis potentially via maintaining mitochondrial homeostasis.

The apoptosis of proximal tubule epithelial cells (PTECs) is a critical event of acute kidney injury (AKI). Tetratricopeptide repeat domain 36 (TTC36) with three tetratricopeptide repeats is evolutionarily conserved across mammals, which functions as a chaperone for heat shock protein 70. We have revealed that TTC36 is specifically expressed in PTECs in our previous work. There are few studies about the role of TTC36 in AKI. In this study, we observed that TTC36 was obviously down-regulated in a mouse model of acute kidney injury established by ischemia/reperfusion and its expression was negatively related to the degree of kidney injury. In addition, TTC36 protected HK2 cells against cisplatin-induced apoptosis. Moreover, we discovered the mechanism that TTC36 mitigated cisplatin-triggered mitochondrial disorder via partially sustaining the membrane potential of mitochondria and mitochondrial autophagy-related gene expression. Collectively, these results suggested that TTC36 plays a protective role in the cisplatin-induced apoptosis of renal tubular cells through maintaining the mitochondrial potential and mitochondrial autophagy-related genes' expression to some extent. These observations highlight the essential role of TTC36 in regulating PTEC apoptosis and imply TTC36/mitochondrial homeostasis axis as a potential target for the therapeutic intervention in AKI.

Truncation of the TPR domain of OGT alters substrate and glycosite selection.

O-GlcNAc transferase (OGT) is an essential enzyme that installs O-linked N-acetylglucosamine (O-GlcNAc) to thousands of protein substrates. OGT and its isoforms select from these substrates through the tetratricopeptide repeat (TPR) domain, yet the impact of truncations to the TPR domain on substrate and glycosite selection is unresolved. Here, we report the effects of iterative truncations to the TPR domain of OGT on substrate and glycosite selection with the model protein GFP-JunB and the surrounding O-GlcNAc proteome in U2OS cells. Iterative truncation of the TPR domain of OGT maintains glycosyltransferase activity but alters subcellular localization of OGT in cells. The glycoproteome and glycosites modified by four OGT TPR isoforms were examined on the whole proteome and a single target protein, GFP-JunB. We found the greatest changes in O-GlcNAc on proteins associated with mRNA splicing processes and that the first four TPRs of the canonical nucleocytoplasmic OGT had the broadest substrate scope. Subsequent glycosite analysis revealed that alteration to the last four TPRs corresponded to the greatest shift in the resulting O-GlcNAc consensus sequence. This dataset provides a foundation to analyze how perturbations to the TPR domain and expression of OGT isoforms affect the glycosylation of substrates, which will be critical for future efforts in protein engineering of OGT, the biology of OGT isoforms, and diseases associated with the TPR domain of OGT.