Sequence-specific Effects Research Articles

Kinetics of Polyampholyte Dimerization: Influence of Charge Sequences

Polyampholytes (PAs) exhibit complex behaviors in various environments influenced by their charge distribution. This study focuses on the kinetics of dimerization of PAs, aiming to elucidate the underlying mechanisms and clarify relevant characteristics of the charge sequence. We focus on PAs with non-zero net charges, employing molecular dynamics simulations and theoretical analyses to examine how charge sequences influence the rates of dimer formation and dissociation. Our findings reveal that the charge sequence of tails and the blockiness of the minority charge group markedly influence the kinetics of dimerization: large blockiness and tails with a high number of majority-type charges slow down the dissociation of dimers. Additionally, the presence of an extended (central) block of the majority charge promotes structural diversity. Within dimer states, blocks alternate between intra- and inter-chain contacts. The duration times in the dimer states are significantly longer than the typical dwell times of block inter-contacts, with a notable extension when multiple blocks are engaged. Intrinsically disordered proteins (IDPs) play crucial roles in cellular functions, primarily due to their ability to undergo rapid conformational changes and form transient complexes. These properties largely depend on the sequence of charged residues. We provide insights into the fundamental principles governing the structural and dynamical properties of polyampholytic IDP, emphasizing the importance of sequence-specific effects on both aggregation and dissociation.

Multimodal Golden DNA Superstructures (GDSs) for Highly Efficient Photothermal Immunotherapy.

DNA-templated metallization has emerged as an efficient strategy for creating nanoscale-metal DNA hybrid structures with a desirable conformation and function. Despite the potential of DNA-metal hybrids, their use as combinatory therapeutic agents has rarely been examined. Herein, we present a simple approach for fabricating a multipurpose DNA superstructure that serves as an efficient photoimmunotherapy agent. Specifically, we adsorb and locally concentrate Au ions onto DNA superstructures through induced local reduction, resulting in the formation of Au nanoclusters. The mechanical and optical properties of these metallic nanoclusters can be rationally controlled by their conformations and metal ions. The resulting golden DNA superstructures (GDSs) exhibit significant photothermal effects that induce cancer cell apoptosis. When sequence-specific immunostimulatory effects of DNA are combined, GDSs provide a synergistic effect to eradicate cancer and inhibit metastasis, demonstrating potential as a combinatory therapeutic agent for tumor treatment. Altogether, the DNA superstructure-templated metal casting system offers promising materials for future biomedical applications.

Mapping the configurational landscape and aggregation phase behavior of the tau protein fragment PHF6.

The PHF6 (Val-Gln-Ile-Val-Tyr-Lys) motif, found in all isoforms of the microtubule-associated protein tau, forms an integral part of ordered cores of amyloid fibrils formed in tauopathies and is thought to play a fundamental role in tau aggregation. Because PHF6 as an isolated hexapeptide assembles into ordered fibrils on its own, it is investigated as a minimal model for insight into the initial stages of aggregation of larger tau fragments. Even for this small peptide, however, the large length and time scales associated with fibrillization pose challenges for simulation studies of its dynamic assembly, equilibrium configurational landscape, and phase behavior. Here, we develop an accurate, bottom-up coarse-grained model of PHF6 for large-scale simulations of its aggregation, which we use to uncover molecular interactions and thermodynamic driving forces governing its assembly. The model, not trained on any explicit information about fibrillar structure, predicts coexistence of formed fibrils with monomers in solution, and we calculate a putative equilibrium phase diagram in concentration-temperature space. We also characterize the configurational and free energetic landscape of PHF6 oligomers. Importantly, we demonstrate with a model of heparin that this widely studied cofactor enhances the aggregation propensity of PHF6 by ordering monomers during nucleation and remaining associated with growing fibrils, consistent with experimentally characterized heparin-tau interactions. Overall, this effort provides detailed molecular insight into PHF6 aggregation thermodynamics and pathways and, furthermore, demonstrates the potential of modern multiscale modeling techniques to produce predictive models of amyloidogenic peptides simultaneously capturing sequence-specific effects and emergent aggregate structures.

Differential effects of conventional and high-definition transcranial direct-current stimulation of the motor cortex on implicit motor sequence learning.

Conventional transcranial direct-current stimulation (tDCS) delivered to the primary motor cortex (M1) has been shown to enhance implicit motor sequence learning (IMSL). Conventional tDCS targets M1 but also the motor association cortices (MAC), making the precise contribution of these areas to IMSL presently unclear. We aimed to address this issue by comparing conventional tDCS of M1 and MAC to 4*1 high-definition (HD) tDCS, which more focally targets M1. In this mixed-factorial, sham-controlled, crossover study in 89 healthy young adults, we used mixed-effects models to analyse sequence-specific and general learning effects in the acquisition and short- and long-term consolidation phases of IMSL, as measured by the serial reaction time task. Conventional tDCS did not influence general learning, improved sequence-specific learning during acquisition (anodal: M= 42.64ms, sham: M= 32.87ms, p =.041), and seemingly deteriorated it at long-term consolidation (anodal: M= 75.37ms, sham: M= 86.63ms, p =.019). HD tDCS did not influence general learning, slowed performance specifically in sequential blocks across all learning phases (all p's < .050), and consequently deteriorated sequence-specific learning during acquisition (anodal: M= 24.13ms, sham: M= 35.67ms, p =.014) and long-term consolidation (anodal: M= 60.03ms, sham: M= 75.01ms, p =.002). Our findings indicate that the observed superior conventional tDCS effects on IMSL are possibly attributable to a generalized stimulation of M1 and/or adjacent MAC, rather than M1 alone. Alternatively, the differential effects can be attributed to cathodal inhibition of other cortical areas involved in IMSL by the 4*1 HD tDCS return electrodes, and/or more variable electric field strengths induced by HD tDCS, compared with conventional tDCS.

Peptide collision cross sections of 22 post-translational modifications

Recent advances have rekindled the interest in ion mobility as an additional dimension of separation in mass spectrometry (MS)-based proteomics. Ion mobility separates ions according to their size and shape in the gas phase. Here, we set out to investigate the effect of 22 different post-translational modifications (PTMs) on the collision cross section (CCS) of peptides. In total, we analyzed ~4300 pairs of matching modified and unmodified peptide ion species by trapped ion mobility spectrometry (TIMS). Linear alignment based on spike-in reference peptides resulted in highly reproducible CCS values with a median coefficient of variation of 0.26%. On a global level, we observed a redistribution in the m/z vs. ion mobility space for modified peptides upon changes in their charge state. Pairwise comparison between modified and unmodified peptides of the same charge state revealed median shifts in CCS between −1.4% (arginine citrullination) and +4.5% (O-GlcNAcylation). In general, increasing modified peptide masses were correlated with higher CCS values, in particular within homologous PTM series. However, investigating the ion populations in more detail, we found that the change in CCS can vary substantially for a given PTM and is partially correlated with the gas phase structure of its unmodified counterpart. In conclusion, our study shows PTM- and sequence-specific effects on the cross section of peptides, which could be further leveraged for proteome-wide PTM analysis.Graphical

The role of environmental contextual cues in sequence learning: evidence from a virtual maze context

Studies on sequence learning usually focus on single, isolated stimuli that are presented sequentially. For example, in the serial reaction time (RT) task, stimuli are either presented in a predictable sequence or in a random sequence, and better performance with the predictable sequence is taken as evidence for sequence-specific learning. Yet, little is known about the role of environmental context cues in sequence learning. If the target stimuli are embedded in a meaningful context, would this facilitate learning by providing helpful contextual associations or would it hinder learning by adding distracting stimuli? This question was examined in two studies. A pilot study compared sequence learning in a virtual maze with a horizontal vs. vertical maze context, in which arrow stimuli guide spatial lever movement responses that resulted in a corresponding virtual transport on the screen. The results showed only overall somewhat better performance with the vertical maze compared to the horizontal maze, but general practice effects and sequence-specific learning effects were the same for both contexts. The main study compared sequence learning with a maze context to sequence learning of arrows without a maze context. The results showed significantly better learning with maze context than without context. These data suggest that the maze context facilitated sequence learning by inducing a meaningful spatial representation (“mental map”) similar to that formed in wayfinding.

Learning a covert sequence of effector movements: limits to its acquisition

Sequence learning in serial reaction time (SRT) tasks is an established, lab-based experimental paradigm to study acquisition and transfer of skills based on the detection of predictable regularities in stimulus and motor response sequences. Participants learn a sequence of targets and responses to these targets by associating the responses with subsequently presented targets. In the traditional paradigm, however, actions and response targets are directly related. In contrast, the present study asked whether participants would demonstrate acquisition of a sequence of effector movements of the left vs. right hand (e.g., hand sequence learning), whilst the actual targets and associated finger responses are unpredictable. Twenty-seven young adults performed a SRT task to visually presented characters with the index or middle fingers of both hands. While the specific fingers to respond with were randomly selected for each target presentation, both hands followed a covert sequence. We asked whether participants would learn the underlying hand sequence as demonstrated by shortened response latencies and increased accuracy compared to a fully randomized hand sequence. The results show sequence-specific learning effects. However, categorization of hand responses depending on the previous response suggested that learning occurred predominantly for subsequent finger responses of the same hand, which added to general hand-based priming. Nevertheless, a marginally significant effect was observed even for predictable shifts between hands when homologous fingers were involved. Our results thus suggest that humans are able to benefit from predictable within-hand finger shifts but less so for predicted shifts between hands.

The Effect of Positive Charge Distribution on the Cryoprotective Activity of Dehydrins.

Dehydrins are intrinsically disordered proteins expressed ubiquitously throughout the plant kingdom in response to desiccation. Dehydrins have been found to have a cryoprotective effect on lactate dehydrogenase (LDH) in vitro, which is in large part influenced by their hydrodynamic radius rather than the order of the amino acids within the sequence (alternatively, this may be a sequence specific effect). However, it seems that a different mechanism may underpin the cryoprotection that they confer to the cold-labile yeast frataxin homolog-1 (Yfh1). Circular dichroism spectroscopy (CD) was used to assess the degree of helicity of Yfh1 at 1 °C, both alone and in the presence of several dehydrin constructs. Three constructs were compared to the wild type: YSK2-K→R (lysine residues substituted with arginine), YSK2-Neutral (locally neutralized charge), and YSK2-SpaceK (evenly distributed positive charge). The results show that sequence rearrangements and minor substitutions have little impact on the ability of the dehydrin to preserve LDH activity. However, when the positive charge of the dehydrin is locally neutralized or evenly distributed, the dehydrin becomes less efficient at promoting structure in Yfh1 at low temperatures. This suggests that a stabilizing, charge-based interaction occurs between dehydrins and Yfh1. Dehydrins are intrinsically disordered proteins, expressed by certain organisms to improve desiccation tolerance. These proteins are thought to serve many cellular roles, such as the stabilization of membranes, DNA, and proteins. However, the molecular mechanisms underlying the function of dehydrins are not well understood. Here, we examine the importance of positive charges in dehydrin sequences by making substitutions and comparing their effects in the cryoprotection of two different proteins.

Homologues not needed: Structure prediction from a protein language model

Accurate protein structure predictors use clusters of homologues, which disregard sequence specific effects. In this issue of Structure, Weißenow and colleagues report a deep learning-based tool, EMBER2, that efficiently predicts the distances in a protein structure from its amino acid sequence only. This approach should enable the analysis of mutation effects.

Reducing Peptide Sequence Bias in Quantitative Mass Spectrometry Data with Machine Learning.

Quantitative mass spectrometry measurements of peptides necessarily incorporate sequence-specific biases that reflect the behavior of the peptide during enzymatic digestion and liquid chromatography and in a mass spectrometer. These sequence-specific effects impair quantification accuracy, yielding peptide quantities that are systematically under- or overestimated. We provide empirical evidence for the existence of such biases, and we use a deep neural network, called Pepper, to automatically identify and reduce these biases. The model generalizes to new proteins and new runs within a related set of tandem mass spectrometry experiments, and the learned coefficients themselves reflect expected physicochemical properties of the corresponding peptide sequences. The resulting adjusted abundance measurements are more correlated with mRNA-based gene expression measurements than the unadjusted measurements. Pepper is suitable for data generated on a variety of mass spectrometry instruments and can be used with labeled or label-free approaches and with data-independent or data-dependent acquisition.

RNA induces unique tau strains and stabilizes Alzheimer’s disease seeds

Tau aggregation underlies neurodegenerative tauopathies, and transcellular propagation of tau assemblies of unique structure, i.e., strains, may underlie the diversity of these disorders. Polyanions have been reported to induce tau aggregation in vitro, but the precise trigger to convert tau from an inert to a seed-competent form in disease states is unknown. RNA triggers tau fibril formation in vitro and has been observed to associate with neurofibrillary tangles in human brain. Here, we have tested whether RNA exerts sequence-specific effects on tau assembly and strain formation. We found that three RNA homopolymers, polyA, polyU, and polyC, all bound tau, but only polyA RNA triggered seed and fibril formation. In addition, polyA:tau seeds and fibrils were sensitive to RNase. We also observed that the origin of the RNA influenced the ability of tau to adopt a structure that would form stable strains. Human RNA potently induced tau seed formation and created tau conformations that preferentially formed stable strains in a HEK293T cell model, whereas RNA from other sources, or heparin, produced strains that were not stably maintained in cultured cells. Finally, we found that soluble, but not insoluble seeds from Alzheimer’s disease brain were also sensitive to RNase. We conclude that human RNA specifically induces formation of stable tau strains and may trigger the formation of dominant pathological assemblies that propagate in Alzheimer’s disease and possibly other tauopathies.

The Specificity of Transgene Suppression in Plants by Exogenous dsRNA.

The phenomenon of RNA interference (RNAi) is widely used to develop new approaches for crop improvement and plant protection. Recent investigations show that it is possible to downregulate plant transgenes, as more prone sequences to silencing than endogenous genes, by exogenous application of double-stranded RNAs (dsRNAs) and small interfering RNAs (siRNAs). However, there are scarce data on the specificity of exogenous RNAs. In this study, we explored whether plant transgene suppression is sequence-specific to exogenous dsRNAs and whether similar effects can be caused by exogenous DNAs that are known to be perceived by plants and induce certain epigenetic and biochemical changes. We treated transgenic plants of Arabidopsis thaliana bearing the neomycin phosphotransferase II (NPTII) transgene with specific synthetic NPTII-dsRNAs and non-specific dsRNAs, encoding enhanced green fluorescent protein (EGFP), as well as with DNA molecules mimicking the applied RNAs. None of the EGFP-dsRNA doses resulted in a significant decrease in NPTII transgene expression in the NPTII-transgenic plants, while the specific NPTII-dsRNA significantly reduced NPTII expression in a dose-dependent manner. Long DNAs mimicking dsRNAs and short DNA oligonucleotides mimicking siRNAs did not exhibit a significant effect on NPTII transgene expression. Thus, exogenous NPTII-dsRNAs induced a sequence-specific and RNA-specific transgene-suppressing effect, supporting external application of dsRNAs as a promising strategy for plant gene regulation.

Implicit learning of perceptual sequences is preserved in Parkinson's disease.

Various studies investigated implicit sequence learning in Parkinson's disease (PD) by means of the traditional motor Serial Reaction Time (SRT) task and found a general pattern of impaired sequence learning. However, as perceptual and motor sequences of the SRT-task were correlated in previous studies, implicit sequential knowledge acquisition that is tested independently from motor sequences remains to be determined in PD. In this study, we investigated implicit sequence learning independently from motor sequence learning in individuals with PD. To this end, we used a perceptual SRT-task that did not rely upon sequential motor knowledge. We measured response times (RTs) of 19 participants with PD (Hoehn & Yahr II or III; mean age 65) and 18 age-matched healthy controls (HC; mean age 61.5) in a perceptual SRT-task. General learning effects and sequence-specific learning effects were analyzed using repeated measures ANOVAs. A significant decreasing linear trend (p < .001) in RTs was revealed in both the PD and HC groups as the SRT-task progressed, indicating general learning effects. Notably, a significant, strong main effect of sequence-specific learning occurred (p < .001), irrespective of group (p = .436). Sequence-specific learning did not differ significantly between the PD (M = 156.5 ms; SD = 50.7) and HC group (M = 173.0 ms; SD = 104.2). Bayesian analyses confirmed this as evidence of absence of an effect (B₁₀ = 3.543). Our results suggest that, at least in Hoehn & Yahr stages II and III, implicit sequential knowledge acquisition may be preserved in individuals with PD, when tested independently from motor sequence learning. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

ROSIGLITAZONE SHOWS SEQUENCE-SPECIFIC SYNERGY WITH CHEMOTHERAPEUTIC DRUGS IN INHIBITION OF MCF-7 BREAST CANCER CELL LINE

Preclinical studies have shown that peroxisome proliferator-activated receptor γ (PPARγ) ligands such as thiazolidinediones (TZDs) can exert antitumor effects against breast cancer and a variety of other cancers. In this study, we investigated the potential of repurposing a PPARγ ligand, rosiglitazone (RGZ), in combination with either of three chemotherapeutic agents, doxorubicin (Dox) or cisplatin (Cis) or 5-fluorouracil (5-FU), for the in-vitro treatment of breast cancer cell line, MCF-7. RGZ augmented the growth inhibition effect of Cis and 5-FU on MCF-7 cells. The synergy was observed only when chemotherapy preceded RGZ and not vice versa, demonstrating a sequence-specific effect. We also observed that the first administered drug gave its cell cycle pattern and apoptosis/necrosis pattern to the subsequently applied drug. Besides, no adipose differentiation in the form of lipid droplet accumulation was induced in treated cells regardless of the used drugs and their sequence of application. Together, our data show a synergistic effect of administering RGZ after Cis or 5-FU and suggests an inhibitory role of RGZ on the chemo-resistant MCF-7 side-populations.

Rosiglitazone Shows Sequence-Specific Synergy with Chemotherapeutic Drugs in Inhibition of MCF-7 Breast Cancer Cell Line

Preclinical studies have shown that peroxisome proliferator-activated receptor γ (PPARγ) ligands such as thiazolidinediones (TZDs) can exert antitumor effects against breast cancer and a variety of other cancers. In this study, we investigated the potential of repurposing a PPARγ ligand, rosiglitazone (RGZ), in combination with either of three chemotherapeutic agents, doxorubicin (Dox) or cisplatin (Cis) or 5-fluorouracil (5-FU), for the in vitro treatment of breast cancer cell line, MCF-7. RGZ augmented the growth inhibition effect of Cis and 5-FU on MCF-7 cells. The synergy was observed only when chemotherapy preceded RGZ and not vice versa, demonstrating a sequence-specific effect. We also observed that the first administered drug gave its cell cycle pattern and apoptosis/necrosis pattern to the subsequently applied drug. Besides, no adipose differentiation in the form of lipid droplet accumulation was induced in treated cells regardless of the used drugs and their sequence of application. Together, our data show a synergistic effect of administering RGZ after Cis or 5-FU and suggests an inhibitory role of RGZ on the chemo-resistant MCF-7 side-populations.

The roles of histone variants in fine-tuning chromatin organization and function.

Histones serve to both package and organize DNA within the nucleus. In addition to histone post-translational modification and chromatin remodelling complexes, histone variants contribute to the complexity of epigenetic regulation of the genome. Histone variants are characterized by a distinct protein sequence and a selection of designated chaperone systems and chromatin remodelling complexes that regulate their localization in the genome. In addition, histone variants can be enriched with specific post-translational modifications, which in turn can provide a scaffold for recruitment of variant-specific interacting proteins to chromatin. Thus, through these properties, histone variants have the capacity to endow specific regions of chromatin with unique character and function in a regulated manner. In this Review, we provide an overview of recent advances in our understanding of the contribution of histone variants to chromatin function in mammalian systems. First, we discuss new molecular insights into chaperone-mediated histone variant deposition. Next, we discuss mechanisms by which histone variants influence chromatin properties such as nucleosome stability and the local chromatin environment both through histone variant sequence-specific effects and through their role in recruiting different chromatin-associated complexes. Finally, we focus on histone variant function in the context of both embryonic development and human disease, specifically developmental syndromes and cancer.

Monomer Sequence Effects on Interfacial Width and Mixing in Self-Assembled Diblock Copolymers

Monomer sequence is shown to directly control interfacial and in-domain mixing in lamellar polystyrene-b-polypeptoid diblock copolymers, where the polypeptoid block is composed of precise sequences of highly segregating (polar) and compatibilizing (nonpolar) repeat units. With monomer-by-monomer sequence control, the effects of blockiness, comonomer distribution, and taper direction on the interfacial width are measured with small-angle X-ray scattering and further understood through simulations with self-consistent field theory. When compatibilizing groups are distributed along the polypeptoid chain, the presence of neighboring polar groups suppresses interfacial mixing and causes the interfacial width to narrow, especially for the blocky sequence. When compatibilizing groups are tapered from the block junction, they are strongly localized at the domain interface and both interfacial mixing and interfacial width are increased. Consequently, tapered sequences produce more pure domain centers, while the distributed materials have compatibilizing groups located throughout the polypeptoid domain, encouraging more polystyrene to mix in. An analogous poly(n-butyl acrylate) system was synthesized to probe the effects of a different interaction parameter (χ). Similar trends with sequence were found but with smaller magnitudes due to the higher χ. This study shows that monomer sequence directly affects segmental mixing both at and away from the interface, with consequences for interfacial width and domain spacing. Along with the sequence-driven nonideal chain conformations shown recently, these combined sequence-specific effects determine the resulting geometry and thermal stability of the self-assembled lamellae, suggesting that comonomer sequence can be used to tailor self-assembling materials without changing the composition or chemistry.

The Dynamics of Hole Transfer in DNA.

High-energy radiation and oxidizing agents can ionize DNA. One electron oxidation gives rise to a radical cation whose charge (hole) can migrate through DNA covering several hundreds of Å, eventually leading to irreversible oxidative damage and consequent disease. Understanding the thermodynamic, kinetic and chemical aspects of the hole transport in DNA is important not only for its biological consequences, but also for assessing the properties of DNA in redox sensing or labeling. Furthermore, due to hole migration, DNA could potentially play an important role in nanoelectronics, by acting as both a template and active component. Herein, we review our work on the dynamics of hole transfer in DNA carried out in the last decade. After retrieving the thermodynamic parameters needed to address the dynamics of hole transfer by voltammetric and spectroscopic experiments and quantum chemical computations, we develop a theoretical methodology which allows for a faithful interpretation of the kinetics of the hole transport in DNA and is also capable of taking into account sequence-specific effects.

Sequence specificity despite intrinsic disorder: How a disease-associated Val/Met polymorphism rearranges tertiary interactions in a long disordered protein.

The role of electrostatic interactions and mutations that change charge states in intrinsically disordered proteins (IDPs) is well-established, but many disease-associated mutations in IDPs are charge-neutral. The Val66Met single nucleotide polymorphism (SNP) in precursor brain-derived neurotrophic factor (BDNF) is one of the earliest SNPs to be associated with neuropsychiatric disorders, and the underlying molecular mechanism is unknown. Here we report on over 250 μs of fully-atomistic, explicit solvent, temperature replica-exchange molecular dynamics (MD) simulations of the 91 residue BDNF prodomain, for both the V66 and M66 sequence. The simulations were able to correctly reproduce the location of both local and non-local secondary structure changes due to the Val66Met mutation, when compared with NMR spectroscopy. We find that the change in local structure is mediated via entropic and sequence specific effects. We developed a hierarchical sequence-based framework for analysis and conceptualization, which first identifies “blobs” of 4-15 residues representing local globular regions or linkers. We use this framework within a novel test for enrichment of higher-order (tertiary) structure in disordered proteins; the size and shape of each blob is extracted from MD simulation of the real protein (RP), and used to parameterize a self-avoiding heterogenous polymer (SAHP). The SAHP version of the BDNF prodomain suggested a protein segmented into three regions, with a central long, highly disordered polyampholyte linker separating two globular regions. This effective segmentation was also observed in full simulations of the RP, but the Val66Met substitution significantly increased interactions across the linker, as well as the number of participating residues. The Val66Met substitution replaces β-bridging between V66 and V94 (on either side of the linker) with specific side-chain interactions between M66 and M95. The protein backbone in the vicinity of M95 is then free to form β-bridges with residues 31-41 near the N-terminus, which condenses the protein. A significant role for Met/Met interactions is consistent with previously-observed non-local effects of the Val66Met SNP, as well as established interactions between the Met66 sequence and a Met-rich receptor that initiates neuronal growth cone retraction.

Sequence-Specific Mucins for Glycocalyx Engineering.

Few approaches exist for the stable and controllable synthesis of customized mucin glycoproteins for glycocalyx editing in eukaryotic cells. Taking advantage of custom gene synthesis and a biology-by-parts approach to cDNA construction, we build a library of swappable DNA bricks for mucin leader tags, membrane anchors, cytoplasmic motifs, and optical reporters, as well as codon-optimized native mucin repeats and newly designed domains for synthetic mucins. We construct a library of over 50 mucins, each with unique chemical, structural, and optical properties and describe how additional permutations could readily be constructed. We apply the library to explore sequence-specific effects on glycosylation for engineering of mucins. We find that the extension of the immature α-GalNAc Tn-antigen to Core 1 and Core 2 glycan structures depends on the underlying peptide backbone sequence. Glycosylation could also be influenced through recycling motifs on the mucin cytoplasmic tail. We expect that the mucin parts inventory presented here can be broadly applied for glycocalyx research and mucin-based biotechnologies.