Quantifying Siglec-sialylated ligand interactions: a versatile 19F-T2 CPMG filtered competitive NMR displacement assay.

Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are integral cell surface proteins crucial for the regulation of immune responses and the maintenance of immune tolerance through interactions with sialic acids. Siglecs recognize sialic acid moieties, usually found at the end of N-glycan and O-glycan chains. However, the different Siglecs prefer diverse presentations of the recognized sialic acid, depending on the type of glycosidic linkage used to link to the contiguous Gal/GalNAc or sialic acid moieties. This fact, together with possible O- or N-substitutions at the recognized glycan epitope significantly influences their roles in various immune-related processes. Understanding the molecular details of Siglec-sialoglycan interactions is essential for unraveling their specificities and for the development of new molecules targeting these receptors. While traditional biophysical methods like isothermal titration calorimetry (ITC) have been utilized to measure binding between lectins and glycans, contemporary techniques such as surface plasmon resonance (SPR), microscale thermophoresis (MST), and biolayer interferometry (BLI) offer improved throughput. However, these methodologies require chemical modification and immobilization of at least one binding partner, which can interfere the recognition between the lectin and the ligand. Since Siglecs display a large range of dissociation constants, depending on the (bio)chemical nature of the interacting partner, a general and robust method that could monitor and quantify binding would be highly welcomed. Herein, we propose the application of an NMR-based a competitive displacement assay, grounded on 19F T2-relaxation NMR and on the design, synthesis, and use of a strategic spy molecule, to assess and quantify sialoside ligand binding to Siglecs. We show that the use of this specific approach allows the quantification of Siglec binding for natural and modified sialosides, multivalent sialosides, and sialylated glycoproteins in solution, which differ in binding affinities in more than two orders of magnitude, thus providing invaluable insights into sialoglycan-mediated interactions.

Potential pandemic risk of circulating swine H1N2 influenza viruses.

Influenza A viruses in swine have considerable genetic diversity and continue to pose a pandemic threat to humans due to a potential lack of population level immunity. Here we describe a pipeline to characterize and triage influenza viruses for their pandemic risk and examine the pandemic potential of two widespread swine origin viruses. Our analysis reveals that a panel of human sera collected from healthy adults in 2020 has no cross-reactive neutralizing antibodies against a α-H1 clade strain (α-swH1N2) but do against a γ-H1 clade strain. The α-swH1N2 virus replicates efficiently in human airway cultures and exhibits phenotypic signatures similar to the human H1N1 pandemic strain from 2009 (H1N1pdm09). Furthermore, α-swH1N2 is capable of efficient airborne transmission to both naïve ferrets and ferrets with prior seasonal influenza immunity. Ferrets with H1N1pdm09 pre-existing immunity show reduced α-swH1N2 viral shedding and less severe disease signs. Despite this, H1N1pdm09-immune ferrets that became infected via the air can still onward transmit α-swH1N2 with an efficiency of 50%. These results indicate that this α-swH1N2 strain has a higher pandemic potential, but a moderate level of impact since there is reduced replication fitness and pathology in animals with prior immunity.

Epistasis mediates the evolution of the receptor binding mode in recent human H3N2 hemagglutinin.

The receptor-binding site of influenza A virus hemagglutinin partially overlaps with major antigenic sites and constantly evolves. In this study, we observe that mutations G186D and D190N in the hemagglutinin receptor-binding site have coevolved in two recent human H3N2 clades. X-ray crystallography results show that these mutations coordinately drive the evolution of the hemagglutinin receptor binding mode. Epistasis between G186D and D190N is further demonstrated by glycan binding and thermostability analyses. Immunization and neutralization experiments using mouse and human samples indicate that the evolution of receptor binding mode is accompanied by a change in antigenicity. Besides, combinatorial mutagenesis reveals that G186D and D190N, along with other natural mutations in recent H3N2 strains, alter the compatibility with a common egg-adaptive mutation in seasonal influenza vaccines. Overall, our findings elucidate the role of epistasis in shaping the recent evolution of human H3N2 hemagglutinin and substantiate the high evolvability of its receptor-binding mode.

Vaccine priming of rare HIV broadly neutralizing antibody precursors in nonhuman primates.

Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.

Heterologous prime-boost vaccination drives early maturation of HIV broadly neutralizing antibody precursors in humanized mice.

A protective HIV vaccine will likely need to induce broadly neutralizing antibodies (bnAbs). Vaccination with the germline-targeting immunogen eOD-GT8 60mer adjuvanted with AS01B was found to induce VRC01-class bnAb precursors in 97% of vaccine recipients in the IAVI G001 phase 1 clinical trial; however, heterologous boost immunizations with antigens more similar to the native glycoprotein will be required to induce bnAbs. Therefore, we designed core-g28v2 60mer, a nanoparticle immunogen to be used as a first boost after eOD-GT8 60mer priming. We found, using a humanized mouse model approximating human conditions of VRC01-class precursor B cell diversity, affinity, and frequency, that both protein- and mRNA-based heterologous prime-boost regimens induced VRC01-class antibodies that gained key mutations and bound to near-native HIV envelope trimers lacking the N276 glycan. We further showed that VRC01-class antibodies induced by mRNA-based regimens could neutralize pseudoviruses lacking the N276 glycan. These results demonstrated that heterologous boosting can drive maturation toward VRC01-class bnAb development and supported the initiation of the IAVI G002 phase 1 trial testing mRNA-encoded nanoparticle prime-boost regimens.

mRNA-LNP HIV-1 trimer boosters elicit precursors to broad neutralizing antibodies.

Germline-targeting (GT) HIV vaccine strategies are predicated on deriving broadly neutralizing antibodies (bnAbs) through multiple boost immunogens. However, as the recruitment of memory B cells (MBCs) to germinal centers (GCs) is inefficient and may be derailed by serum antibody-induced epitope masking, driving further B cell receptor (BCR) modification in GC-experienced B cells after boosting poses a challenge. Using humanized immunoglobulin knockin mice, we found that GT protein trimer immunogen N332-GT5 could prime inferred-germline precursors to the V3-glycan-targeted bnAb BG18 and that B cells primed by N332-GT5 were effectively boosted by either of two novel protein immunogens designed to have minimum cross-reactivity with the off-target V1-binding responses. The delivery of the prime and boost immunogens as messenger RNA lipid nanoparticles (mRNA-LNPs) generated long-lasting GCs, somatic hypermutation, and affinity maturation and may be an effective tool in HIV vaccine development.

Mental Health Education and Utilization Among Patients with Vestibular Disorders.

To explore the receipt of mental health education, assessment, and referrals, and mental health service use among individuals with vestibular disorders. Patients with vestibular disorders living in the US, Australia, Canada, and the UK were surveyed through social media forums. Questionnaires assessed demographics, anxiety (Generalized Anxiety Disorder-7), depression (Center for Epidemiological Studies Depression-10), dizziness (Dizziness Handicap Inventory), and type of professional providing mental health education, assessment, referral, and treatment. The 226 participants were largely White (90%), educated (67% holding an associate's degree or higher) women (88%) with an average age of 45 who self-identified as having chronic vestibular symptoms (78%), as opposed to episodic ones (22%). Fifty-two percent reported never receiving verbal education, written education (69%), mental health assessment (54%), or referral (72%). Participants were more likely to receive mental health treatment in the past if they had received verbal resources and/or referrals from clinicians. The majority of patients with vestibular disorders report that medical professionals have not provided education, mental health assessment, or a mental health referral.

Rules of engagement for condensins and cohesins guide mitotic chromosome formation.

During mitosis, interphase chromatin is rapidly converted into rod-shaped mitotic chromosomes. Using Hi-C, imaging, proteomics and polymer modeling, we determine how the activity and interplay between loop-extruding SMC motors accomplishes this dramatic transition. Our work reveals rules of engagement for SMC complexes that are critical for allowing cells to refold interphase chromatin into mitotic chromosomes. We find that condensin disassembles interphase chromatin loop organization by evicting or displacing extrusive cohesin. In contrast, condensin bypasses cohesive cohesins, thereby maintaining sister chromatid cohesion while separating the sisters. Studies of mitotic chromosomes formed by cohesin, condensin II and condensin I alone or in combination allow us to develop new models of mitotic chromosome conformation. In these models, loops are consecutive and not overlapping, implying that condensins do not freely pass one another but stall upon encountering each other. The dynamics of Hi-C interactions and chromosome morphology reveal that during prophase loops are extruded in vivo at ~1-3 kb/sec by condensins as they form a disordered discontinuous helical scaffold within individual chromatids.

Cross-species spill-over potential of the H9N2 bat influenza A virus.

In 2017, a novel influenza A virus (IAV) was isolated from an Egyptian fruit bat. In contrast to other bat influenza viruses, the virus was related to avian A(H9N2) viruses and was probably the result of a bird-to-bat transmission event. To determine the cross-species spill-over potential, we biologically characterize features of A/bat/Egypt/381OP/2017(H9N2). The virus has a pH inactivation profile and neuraminidase activity similar to those of human-adapted IAVs. Despite the virus having an avian virus-like preference for α2,3 sialic acid receptors, it is unable to replicate in male mallard ducks; however, it readily infects ex-vivo human respiratory cell cultures and replicates in the lungs of female mice. A/bat/Egypt/381OP/2017 replicates in the upper respiratory tract of experimentally-infected male ferrets featuring direct-contact and airborne transmission. These data suggest that the bat A(H9N2) virus has features associated with increased risk to humans without a shift to a preference for α2,6 sialic acid receptors.

Targeting inhibitory Siglec-3 to suppress IgE-mediated human basophil degranulation.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin-3 (Siglec-3 [CD33]) is a major Siglec expressed on human mast cells and basophils; engagement of CD33 leads to inhibition of cellular signaling via immunoreceptor tyrosine-based inhibitory motifs. OBJECTIVE: We sought to inhibit human basophil degranulation by simultaneously recruiting inhibitory CD33 to the IgE-FcεRI complex by using monoclonal anti-IgE directly conjugated to CD33 ligand (CD33L). METHODS: Direct and indirect basophil activation tests (BATs) were used to assess both antigen-specific (peanut) and antigen-nonspecific (polyclonal anti-IgE) stimulation. Whole blood from donors with allergy was used for direct BAT, whereas blood from donors with nonfood allergy was passively sensitized with plasma from donors with peanut allergy in the indirect BAT. Blood was incubated with anti-IgE-CD33L or controls for 1 hour or overnight and then stimulated with peanut, polyclonal anti-IgE, or N-formylmethionyl-leucyl-phenylalanine for 30 minutes. Degranulation was determined by measuring CD63 expression on the basophil surface by flow cytometry. RESULTS: Incubation for 1 hour with anti-IgE-CD33L significantly reduced basophil degranulation after both allergen-induced (peanut) and polyclonal anti-IgE stimulation, with further suppression after overnight incubation with anti-IgE-CD33L. As expected, anti-IgE-CD33L did not block basophil degranulation due to N-formylmethionyl-leucyl-phenylalanine, providing evidence that this inhibition is IgE pathway-specific. Finally, CD33L is necessary for this suppression, as monoclonal anti-IgE without CD33L was unable to reduce basophil degranulation. CONCLUSIONS: Pretreating human basophils with anti-IgE-CD33L significantly suppressed basophil degranulation through the IgE-FcεRI complex. The ability to abrogate IgE-mediated basophil degranulation is of particular interest, as treatment with anti-IgE-CD33L before antigen exposure could have broad implications for the treatment of food, drug, and environmental allergies.

Evolution of human H3N2 influenza virus receptor specificity has substantially expanded the receptor-binding domain site.

Hemagglutinins (HAs) from human influenza viruses descend from avian progenitors that bind α2-3-linked sialosides and must adapt to glycans with α2-6-linked sialic acids on human airway cells to transmit within the human population. Since their introduction during the 1968 pandemic, H3N2 viruses have evolved over the past five decades to preferentially recognize human α2-6-sialoside receptors that are elongated through addition of poly-LacNAc. We show that more recent H3N2 viruses now make increasingly complex interactions with elongated receptors while continuously selecting for strains maintaining this phenotype. This change in receptor engagement is accompanied by an extension of the traditional receptor-binding site to include residues in key antigenic sites on the surface of HA trimers. These results help explain the propensity for selection of antigenic variants, leading to vaccine mismatching, when H3N2 viruses are propagated in chicken eggs or cells that do not contain such receptors.

Unraveling Molecular Recognition of Glycan Ligands by Siglec-9 via NMR Spectroscopy and Molecular Dynamics Modeling.

Human sialic-acid-binding immunoglobulin-like lectin-9 (Siglec-9) is a glycoimmune checkpoint receptor expressed on several immune cells. Binding of Siglec-9 to sialic acid containing glycans (sialoglycans) is well documented to modulate its functions as an inhibitory receptor. Here, we first assigned the amino acid backbone of the Siglec-9 V-set domain (Siglec-9d1), using well-established triple resonance three-dimensional nuclear magnetic resonance (NMR) methods. Then, we combined solution NMR and molecular dynamic simulation methods to decipher the molecular details of the interaction of Siglec-9 with the natural ligands α2,3 and α2,6 sialyl lactosamines (SLN), sialyl Lewis X (sLeX), and 6-O sulfated sLeX and with two synthetically modified sialoglycans that bind with high affinity. As expected, Neu5Ac is accommodated between the F and G ß-strands at the canonical sialic acid binding site. Addition of a heteroaromatic scaffold 9N-5-(2-methylthiazol-4-yl)thiophene sulfonamide (MTTS) at the C9 position of Neu5Ac generates new interactions with the hydrophobic residues located at the G-G' loop and the N-terminal region of Siglec-9. Similarly, the addition of the aromatic substituent (5-N-(1-benzhydryl-1H-1,2,3-triazol-4-yl)methyl (BTC)) at the C5 position of Neu5Ac stabilizes the conformation of the long and flexible B'-C loop present in Siglec-9. These results expose the underlying mechanism responsible for the enhanced affinity and specificity for Siglec-9 for these two modified sialoglycans and sheds light on the rational design of the next generation of modified sialoglycans targeting Siglec-9.

Identifying general reaction conditions by bandit optimization.

Reaction conditions that are generally applicable to a wide variety of substrates are highly desired, especially in the pharmaceutical and chemical industries1-6. Although many approaches are available to evaluate the general applicability of developed conditions, a universal approach to efficiently discover these conditions during optimizations is rare. Here we report the design, implementation and application of reinforcement learning bandit optimization models7-10 to identify generally applicable conditions by efficient condition sampling and evaluation of experimental feedback. Performance benchmarking on existing datasets statistically showed high accuracies for identifying general conditions, with up to 31% improvement over baselines that mimic state-of-the-art optimization approaches. A palladium-catalysed imidazole C-H arylation reaction, an aniline amide coupling reaction and a phenol alkylation reaction were investigated experimentally to evaluate use cases and functionalities of the bandit optimization model in practice. In all three cases, the reaction conditions that were most generally applicable yet not well studied for the respective reaction were identified after surveying less than 15% of the expert-designed reaction space.

Glyco-engineered MDCK cells display preferred receptors of H3N2 influenza absent in eggs used for vaccines.

Evolution of human H3N2 influenza viruses driven by immune selection has narrowed the receptor specificity of the hemagglutinin (HA) to a restricted subset of human-type (Neu5Acα2-6 Gal) glycan receptors that have extended poly-LacNAc (Galß1-4GlcNAc) repeats. This altered specificity has presented challenges for hemagglutination assays, growth in laboratory hosts, and vaccine production in eggs. To assess the impact of extended glycan receptors on virus binding, infection, and growth, we have engineered N-glycan extended (NExt) cell lines by overexpressing ß3-Ν-acetylglucosaminyltransferase 2 in MDCK, SIAT, and hCK cell lines. Of these, SIAT-NExt cells exhibit markedly increased binding of H3 HAs and susceptibility to infection by recent H3N2 virus strains, but without impacting final virus titers. Glycome analysis of these cell lines and allantoic and amniotic egg membranes provide insights into the importance of extended glycan receptors for growth of recent H3N2 viruses and relevance to their production for cell- and egg-based vaccines.

Analog-sensitive Cdk1 as a tool to study mitotic exit: protein phosphatase 1 is required downstream from Cdk1 inactivation in budding yeast.

We show that specific inactivation of the protein kinase Cdk1/cyclin B (Cdc28/Clb2) triggers exit from mitosis in the budding yeast Saccharomyces cerevisiae. Cells carrying the allele cdc28-as1, which makes Cdk1 (Cdc28) uniquely sensitive to the ATP analog 1NM-PP1, were arrested with spindle poisons and then treated with 1NM-PP1 to inhibit Cdk1. This caused the cells to leave mitosis and enter G1-phase as shown by initiation of rebudding (without cytokinesis), induction of mating projections ("shmoos") by α-factor, stabilization of Sic1, and degradation of Clb2. It is known that Cdk1 must be inactivated for cells to exit mitosis, but our results show that inactivation of Cdk1 is not only necessary but also sufficient to initiate the transition from mitosis to G1-phase. This result suggests a system in which to test requirements for particular gene products downstream from Cdk1 inactivation, for example, by combining cdc28-as1 with conditional mutations in the genes of interest. Using this approach, we demonstrate that protein phosphatase 1 (PPase1; Glc7 in S. cerevisiae) is required for mitotic exit and reestablishment of interphase following Cdk1 inactivation. This system could be used to test the need for other protein phosphatases downstream from Cdk1 inactivation, such as PPase 2A and Cdc14, and it could be combined with phosphoproteomics to gain information about the substrates that the various phosphatases act upon during mitotic exit.

My Baby, My Move+: feasibility of a community prenatal wellbeing intervention.

BACKGROUND: Excessive gestational weight gain (EGWG), insufficient prenatal physical activity and sleep, and poor psychological wellbeing independently increase risks for adverse maternal and infant outcomes. A novel approach to mitigate these risks is utilizing peer support in a community-based prenatal intervention. This study assessed the feasibility (acceptability, demand, implementation, and practicality) of a remotely delivered prenatal physical activity intervention called My Baby, My Move + (MBMM +) that aims to increase prenatal physical activity, enhance mood and sleep hygiene, and reduce EGWG. METHODS: Participants were recruited through community organizations, local clinics, and social media platforms in the Fall of 2020 and Spring of 2021. Eligible pregnant women were randomized to either the MBMM + intervention or the control group. Each group met over Zoom for 16 sessions (twice weekly for 60 min over 8 weeks) to learn either behavioral change and wellbeing knowledge and skills (MBMM +) or knowledge and skills related to parenting (control group). Multiple methods of evaluation to better understand the feasibility of the intervention were conducted. RESULTS: A total of 49 women (25 MBMM + intervention, 24 control) completed both pre- and post-survey assessments and were included in the analyses. A subsample of 19 (39%) intervention participants completed a combination of semi-structured interviews/surveys to assess acceptability, demand, implementation, and practicality. Participants expressed positive feedback regarding acceptability (satisfaction and intent to continue use) and were extremely likely or likely to recommend the program to a friend (demand). Implementation metrics were assessed by observation and feedback forms completed by peer leaders and demonstrated high-quality control. Findings suggest that the intervention was practical due to remote sessions and cost-effectiveness. CONCLUSION: The MBMM + intervention was deemed to be a feasible intervention with high acceptability, demand, implementation, and practicality. These findings can be used to inform the scalability of the intervention and implementation of a larger efficacy trial. TRIAL REGISTRATION: 19-1366, initial date is on January 23, 2020.

Evolving spike-protein N-glycosylation in SARS-CoV-2 variants.

Since >3 years, SARS-CoV-2 has plunged humans into a colossal pandemic. Henceforth, multiple waves of infection have swept through the human population, led by variants that were able to partially evade acquired immunity. The co-evolution of SARS-CoV-2 variants with human immunity provides an excellent opportunity to study the interaction between viral pathogens and their human hosts. The heavily N-glycosylated spike-protein of SARS-CoV-2 plays a pivotal role in initiating infection and is the target for host immune-response, both of which are impacted by host-installed N-glycans. Using highly-sensitive DeGlyPHER approach, we compared the N-glycan landscape on spikes of the SARS-CoV-2 Wuhan-Hu-1 strain to seven WHO-defined variants of concern/interest, using recombinantly expressed, soluble spike-protein trimers, sharing same stabilizing-mutations. We found that N-glycan processing is conserved at most sites. However, in multiple variants, processing of N-glycans from high mannose- to complex-type is reduced at sites N165, N343 and N616, implicated in spike-protein function.

Exit from Mitosis in Budding Yeast: Protein Phosphatase 1 is Required Downstream from Cdk1 Inactivation.

We show that inactivation of the protein kinase Cdk1/Cyclin B (Cdc28/Clb 2 in the budding yeast Saccharomyces cerevisiae ) is not only necessary for cells to leave mitosis, as is well known, but also sufficient to trigger mitotic exit. Cells carrying the mutation cdc28-as1 , which makes Cdc28 (Cdk1) uniquely sensitive to the ATP analog 1NM-PP1, were arrested with spindle poisons and then treated with 1NM-PP1 to inhibit Cdk1. This treatment caused the cells to exit mitosis and enter G1-phase as shown by initiation of rebudding (without cytokinesis), production of "shmoos" (when α-factor was present), stabilization of Sic1, and degradation of Clb2. This result provides a system in which to test whether particular gene products are required downstream from Cdk1 inactivation in exit from mitosis. In this system, the mutation cdc28-as1 is combined with a conditional mutation in the gene of interest. Using this approach, we demonstrate that Protein Phosphatase 1 (PPase1; Glc7 in S. cerevisiae ) is required for reestablishment of G1-phase following Cdk1 inactivation. This system could be used to test whether other protein phosphatases are also needed downstream from Cdk1 inactivation, and it could be combined with phosphoproteomics to gain information about the substrates those phosphatases act on during mitotic exit.

Revealing the Specificity of Human H1 Influenza A Viruses to Complex N-Glycans.

Influenza virus infection remains a threat to human health since viral hemagglutinins are constantly drifting, escaping infection and vaccine-induced antibody responses. Viral hemagglutinins from different viruses display variability in glycan recognition. In this context, recent H3N2 viruses have specificity for α2,6 sialylated branched N-glycans with at least three N-acetyllactosamine units (tri-LacNAc). In this work, we combined glycan arrays and tissue binding analyses with nuclear magnetic resonance experiments to characterize the glycan specificity of a family of H1 variants, including the one responsible for the 2009 pandemic outbreak. We also analyzed one engineered H6N1 mutant to understand if the preference for tri-LacNAc motifs could be a general trend in human-type receptor-adapted viruses. In addition, we developed a new NMR approach to perform competition experiments between glycans with similar compositions and different lengths. Our results point out that pandemic H1 viruses differ from previous seasonal H1 viruses by a strict preference for a minimum of di-LacNAc structural motifs.

DeGlyPHER: Highly sensitive site-specific analysis of N-linked glycans on proteins.

Traditional mass spectrometry-based glycoproteomic approaches have been widely used for site-specific N-glycoform analysis, but a large amount of starting material is needed to obtain sampling that is representative of the vast diversity of N-glycans on glycoproteins. These methods also often include a complicated workflow and very challenging data analysis. These limitations have prevented glycoproteomics from being adapted to high-throughput platforms, and the sensitivity of the analysis is currently inadequate for elucidating N-glycan heterogeneity in clinical samples. Heavily glycosylated spike proteins of enveloped viruses, recombinantly expressed as potential vaccines, are prime targets for glycoproteomic analysis. Since the immunogenicity of spike proteins may be impacted by their glycosylation patterns, site-specific analysis of N-glycoforms provides critical information for vaccine design. Using recombinantly expressed soluble HIV Env trimer, we describe DeGlyPHER, a modification of our previously reported sequential deglycosylation strategy to yield a "single-pot" process. DeGlyPHER is an ultrasensitive, simple, rapid, robust, and efficient approach for site-specific analysis of protein N-glycoforms, that we developed for analysis of limited quantities of glycoproteins.