Multiscale approach to the characterization of the interfacial properties of micellar casein and whey protein blends and their effects on recombined dairy creams.

In this study, whipped cream with blends of micellar casein (MCN) and whey protein (WPI) in different ratios were prepared to investigate the role of protein interfacial behavior in determining foam properties at multiple scales, using theoretical modeling, and microscopic and macroscopic analysis. Fluid force microscopy has been used for the first time as a more realistic and direct means of analyzing interfaces properties in multiphase systems. The adsorption kinetics showed that the interfacial permeability constant of WPI (4.24 × 10-4 s-1) was significantly higher than that of the MCN (2.97 × 10-4 s-1), and the WPI interfacial layer had a higher modulus of elasticity (71.38 mN/m) than that of the MCN (47.89 mN/m). This model was validated via the mechanical analysis of the fat globules in real emulsions. The WPI-stabilized fat globule was found to have a higher Young's modulus (219.67 Pa), which contributes to the integrity of its fat globule morphology. As the ratio of MCN was increased in the sample, however, both the interfacial modulus and Young's modulus decreased. Moreover, the rate of partial coalescence was found to increase, a phenomenon that decreased the stability of the emulsion and increased the rate of aeration. The mechanical analysis also revealed a higher level of adhesion between MCN-stabilized fat globule (25.16 nN), which increased fat globule aggregation and emulsion viscosity, while improving thixotropic recovery. The synergistic effect of the blended MCN and WPI provided the highest overrun, at 194.53 %. These studies elucidate the role of the interfacial behavior of proteins in determining the quality of whipped cream and provide ideas for the application of proteins in multiphase systems.

Role of ulinastatin in steroid-induced pancreatitis.

Steroid-induced acute pancreatitis is a rare form of pancreatitis that requires intensive care and has a high morbidity and mortality rate as there is no specific treatment. Management of steroid-induced pancreatitis is generally non-specific and supportive. Here, we are presenting a man in his 40s presented with epigastric pain, fever and vomiting. The patient was diagnosed case of rheumatoid arthritis, for which he was receiving regular 5 mg oral prednisolone therapy. Based on history, and clinical, biochemical and radiological imaging a diagnosis of steroid-induced pancreatitis was made, which was successfully managed with the help of ulinastatin and other supportive treatments. A serine protease inhibitor like ulinastatin may be used early in the clinical management of steroid-induced pancreatitis.

The human alpha7 nicotinic acetylcholine receptor is a host target for the rabies virus glycoprotein.

The rabies virus enters the nervous system by interacting with several molecular targets on host cells to modify behavior and trigger receptor-mediated endocytosis of the virion by poorly understood mechanisms. The rabies virus glycoprotein (RVG) interacts with the muscle acetylcholine receptor and the neuronal α4ß2 subtype of the nicotinic acetylcholine receptor (nAChR) family by the putative neurotoxin-like motif. Given that the neurotoxin-like motif is highly homologous to the α7 nAChR subtype selective snake toxin α-bungarotoxin (αBTX), other nAChR subtypes are likely involved. The purpose of this study is to determine the activity of the RVG neurotoxin-like motif on nAChR subtypes that are expressed in brain regions involved in rabid animal behavior. nAChRs were expressed in Xenopus laevis oocytes, and two-electrode voltage clamp electrophysiology was used to collect concentration-response data to measure the functional effects. The RVG peptide preferentially and completely inhibits α7 nAChR ACh-induced currents by a competitive antagonist mechanism. Tested heteromeric nAChRs are also inhibited, but to a lesser extent than the α7 subtype. Residues of the RVG peptide with high sequence homology to αBTX and other neurotoxins were substituted with alanine. Altered RVG neurotoxin-like peptides showed that residues phenylalanine 192, arginine 196, and arginine 199 are important determinants of RVG peptide apparent potency on α7 nAChRs, while serine 195 is not. The evaluation of the rabies ectodomain reaffirmed the observations made with the RVG peptide, illustrating a significant inhibitory impact on α7 nAChR with potency in the nanomolar range. In a mammalian cell culture model of neurons, we confirm that the RVG peptide binds preferentially to cells expressing the α7 nAChR. Defining the activity of the RVG peptide on nAChRs expands our understanding of basic mechanisms in host-pathogen interactions that result in neurological disorders.

Bispecific antibodies targeting two glycoproteins on SFTSV exhibit synergistic neutralization and protection in a mouse model.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with a high fatality rate of up to 30% caused by SFTS virus (SFTSV). However, no specific vaccine or antiviral therapy has been approved for clinical use. To develop an effective treatment, we isolated a panel of human monoclonal antibodies (mAbs). SF5 and SF83 are two neutralizing mAbs that recognize two viral glycoproteins (Gn and Gc), respectively. We found that their epitopes are closely located, and we then engineered them as several bispecific antibodies (bsAbs). Neutralization and animal experiments indicated that bsAbs display more potent protective effects than the parental mAbs, and the cryoelectron microscopy structure of a bsAb3 Fab-Gn-Gc complex elucidated the mechanism of protection. In vivo virus passage in the presence of antibodies indicated that two bsAbs resulted in less selective pressure and could efficiently bind to all single parental mAb-escape mutants. Furthermore, epitope analysis of the protective mAbs against SFTSV and RVFV indicated that they are all located on the Gn subdomain I, where may be the hot spots in the phleboviruses. Collectively, these data provide potential therapeutic agents and molecular basis for the rational design of vaccines against SFTSV infection.

Alterations of mannosylated glycopatterns recognized by Hippeastrum hybrid lectin in saliva of patients with lung cancer.

OBJECTIVES: Lung cancer (LC) is the malignant tumor with the highest mortality rate worldwide, and precise early diagnosis can improve patient prognosis. The purpose of this study was to investigate whether alterations in the glycopatterns recognized by the Hippeastrum hybrid lectin (HHL) in salivary proteins are associated with the development of LC. MATERIALS AND METHODS: First, we collected saliva samples from LC (15 lung adenocarcinoma (ADC); 15 squamous cell carcinoma (SCC); 15 small cell lung cancer (SCLC)) and 15 benign pulmonary disease (BPD) for high-throughput detection of abundance levels of HHL-recognized glycopatterns using protein microarrays, and then validated the pooled samples from each group with lectin blotting analysis. Finally, the N-glycan profiles of salivary glycoproteins isolated from the pooled samples using HHL-magnetic particle conjugates were characterized separately using MALDI-TOF/TOF-MS. RESULTS: The results showed that the abundance level of glycopatterns recognized by HHL in salivary proteins was elevated in LC compared to BPD. The proportion of mannosylated N-glycans was notably higher in ADC (31.7%), SCC (39.0%), and SCLC (46.6%) compared to BPD (23.3%). CONCLUSIONS: The altered salivary glycopatterns such as oligomannose, Manα1-3Man, or Manα1-6Man N-glycans recognized by HHL might serve as potential biomarkers for the diagnosis of LC patients. CLINICAL RELEVANCE: This study provides crucial information for studying changes in salivary to differentiate between BPD and LC and facilitate the discovery of biomarkers for LC diagnosis based on precise alterations of mannosylated N-glycans in saliva.

CTRP 9 mitigates the apoptosis and unfolded protein response of OGD/R-induced retinal ganglion cells by regulating the AMPK pathway.

C1q/TNF-related protein-9 (CTRP9) has been reported to play roles in several types of retinal diseases. However, the role and the potential mechanism of CTRP9 in glaucoma are still incompletely understood. The expression of CTRP9 in OGD/R-induced retinal ganglion cells (RGCs) was detected by quantitative real-time polymerase chain reaction and western blot assay. Cell proliferation was identified by cell counting Kit-8 assay. Flow cytometry, enzyme-linked immunosorbent assay and western blot assay were performed to assess cell apoptosis. Unfolded protein response (UPR), endoplasmic reticulum (ER) stress and the AMPK pathway were evaluated by western blot assay. The data showed that the expression of CTRP9 was significantly downregulated in OGD/R-induced 661W cells. OGD/R treatment reduced cell viability, promoted cell apoptosis and activated the UPR and ER stress. The overexpression of CTRP9 reversed the effects of OGD/R on 661W cell viability, apoptosis, the UPR and ER stress, as well as the AMPK pathway. However, Compound C, an inhibitor of AMPK signaling, reversed the protection of CTRP9 overexpression against injury from OGD/R in 661W cells. In summary, the results revealed that CTRP9 abated the apoptosis and UPR of OGD/R-induced RGCs by regulating the AMPK pathway, which may provide a promising target for the treatment of glaucoma.

Droplet Microfluidics with Capillary Electrophoresis for Glycan Biomarker Analysis.

Several glycoproteins are validated biomarkers of various diseases such as cancer, cardiovascular diseases, chronic alcohol abuse, or congenital disorders of glycosylation (CDG). In particular, CDG represent a group of more than 150 inherited diseases with varied symptoms affecting multiple organs. The distribution of glycans from target glycoprotein(s) can be used to extract information to help the diagnosis and possibly differentiate subtypes of CDG. Indeed, depending on the glycans and the proteins to which they are attached, glycans can play a very broad range of roles in both physical and biological properties of glycoproteins. For glycans in general, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) has become a staple. Analysis of glycans with CE-LIF requires several sample preparation steps, including release of glycans from the target glycoprotein, fluorescent labeling of glycans, and purification of labeled glycans. Here, we describe the protocol for glycan sample treatment in a microfluidic droplet system prior to CE-LIF of labeled glycans. The microfluidic droplet approach offers full automation, sample, and reagent volume reduction and elimination of contamination from external environment.

The importance of leucine-rich α-2 glycoprotein-1 in acute calculus cholecystitis.

OBJECTIVE: The aim of this prospective, single-center cohort study was to analyze serum leucine-rich α-2-glycoprotein-1 (LRG1) expression in patients with acute cholecystitis (AC) and to investigate its variation depending on symptom duration. PATIENTS AND METHODS: Participants were divided into patients with AC and a healthy control group. At the time of diagnosis, blood samples were collected, and symptom onset times were questioned. Collected serum LRG1 levels were measured. RESULTS: 30 patients and 30 healthy volunteers were included in the study. LRG1 (p=0.008), white blood cells (WBC) (p<0.001), platelet (p=0.003), neutrophil (p<0.001), lymphocyte (p=0.001), and CRP (p=0.014) were significantly different in AC patients vs. the control group. When the correlations of serum laboratory values with the time of onset of symptoms were compared, LRG1 (p<0.001) was significantly correlated, while no significant correlation was observed in C-reactive protein (CRP) (p=0.572), WBC (p=0.155), and neutrophil (p=0.155). CONCLUSIONS: LRG1 expression increases after 24 hours in AC patients. Due to its correlation with symptom duration, we believe it can be helpful for timing cholecystectomy.

Integrated proteomic, phosphoproteomic, and N-glycoproteomic analyses of small extracellular vesicles from C2C12 myoblasts identify specific PTM patterns in ligand-receptor interactions.

Small extracellular vesicles (sEVs) are important mediators of intercellular communication by transferring of functional components (proteins, RNAs, and lipids) to recipient cells. Some PTMs, including phosphorylation and N-glycosylation, have been reported to play important role in EV biology, such as biogenesis, protein sorting and uptake of sEVs. MS-based proteomic technology has been applied to identify proteins and PTM modifications in sEVs. Previous proteomic studies of sEVs from C2C12 myoblasts, an important skeletal muscle cell line, focused on identification of proteins, but no PTM information on sEVs proteins is available.In this study, we systematically analyzed the proteome, phosphoproteome, and N-glycoproteome of sEVs from C2C12 myoblasts with LC-MS/MS. In-depth analyses of the three proteomic datasets revealed that the three proteomes identified different catalogues of proteins, and PTMomic analysis could expand the identification of cargos in sEVs. At the proteomic level, a high percentage of membrane proteins, especially tetraspanins, was identified. The sEVs-derived phosphoproteome had a remarkably high level of tyrosine-phosphorylated sites. The tyrosine-phosphorylated proteins might be involved with EPH-Ephrin signaling pathway. At the level of N-glycoproteomics, several glycoforms, such as complex N-linked glycans and sialic acids on glycans, were enriched in sEVs. Retrieving of the ligand-receptor interaction in sEVs revealed that extracellular matrix (ECM) and cell adhesion molecule (CAM) represented the most abundant ligand-receptor pairs in sEVs. Mapping the PTM information on the ligands and receptors revealed that N-glycosylation mainly occurred on ECM and CAM proteins, while phosphorylation occurred on different categories of receptors and ligands. A comprehensive PTM map of ECM-receptor interaction and their components is also provided.In summary, we conducted a comprehensive proteomic and PTMomic analysis of sEVs of C2C12 myoblasts. Integrated proteomic, phosphoproteomic, and N-glycoproteomic analysis of sEVs might provide some insights about their specific uptake mechanism.

Proteomic and phosphoproteomic reveal immune-related function of milk fat globule membrane in bovine milk of different lactation periods.

Information regarding protein expression and phosphorylation modifications in the bovine milk fat globule membrane is scarce, particularly throughout various lactation periods. This study employed a complete proteome and phosphoproteome between bovine colostrum and mature milk. A total of 11 proteins were seen in both protein expression and phosphorylation levels. There were 400 proteins identified in only protein expression, and 104 phosphoproteins identified in only phosphorylation levels. A total of 232 significant protein characteristics were identified within the proteome and significant phosphorylation sites within 86 phosphoproteins of the phosphoproteome. Biological activities and pathways primarily exhibited associations with the immune system. Simultaneously, a comprehensive analysis of proteins and phosphorylation sites using a multi-omics approach. Hence, the data we have obtained has the potential to expand our understanding of how the bovine milk fat globule membrane might be utilized as a beneficial component in dairy products.

Effect of degalactosylated bovine glycoprotein formulations MAF and M сapsules on lymphopenia and clinical outcomes in hospitalized COVID-19 patients: a randomized clinical trial.

BACKGROUND: Targeting mucosal immunity of the gut, which is known to provide antigen processing, while avoiding excessive or unnecessary inflammation, was tested as a way to modulate COVID-19 severity. METHODS: Randomized open-label trial in 204 adults hospitalized with non-critical COVID-19 who received for 14 days in addition to standard of care (SOC) degalactosylated bovine glycoproteins formulations of either MAF capsules (MAF group) or M capsules (M group) or SOC only (control group). RESULTS: Median recovery time when patients did not require supplemental oxygen was 6 days in both study groups compared to 9 days in the control (MAF vs. control; P = 0.020 and M vs. control; P = 0.004). A greater reduction in mortality was seen in the MAF group compared to the control by day 14 (8.3% vs. 1.6%; P = 0.121) and by day 29 (15.3% vs. 3.2%; P = 0.020), and similarly in the M group by day 14 (8.3% vs. 2.9%; P = 0.276) and by day 29 (15.3% vs. 2.9%; P = 0.017). The proportion of those who had baseline absolute lymphocyte count (ALC) lower than 0.8 × 109/L was 13/63 (20.6%), 17/69 (24.6%), and 18/72 (25.0%) of patients in MAF, M, and control group respectively. Day 29 mortality among these lymphopenic patients was three times higher than for the intent-to-treat population (21% vs. 7%) and consisted in above subgroups: 2/13 (15%), 2/17 (12%), and 6/18 (33%) of patients. The decreased mortality in both study subgroups correlated with greater ALC restoration above 0.8 × 109/L level seen on day 14 in 91% (11/12) and 87.5% (14/16) of survivors in MAF and M subgroups respectively compared to 53.3% (8/15) of survivors in control subgroup. Incidences of any ALC decrease below the baseline level on day 14 occurred in 25.4% of patients in the MAF group and 29.0% of patients in the M group compared to 45.8% in control and ALC depletion by ≥ 50% from the baseline level consisted of 7.9%, 5.8%, and 15.3% of cases in these groups respectively. CONCLUSION: This study showed that both study agents prevented ALC depletion and accelerated its restoration, which is believed to be one of the mechanisms of improved crucial clinical outcomes in hospitalized COVID-19 patients. TRIAL REGISTRATION: The trial was registered after the trial start in ClinicalTrials.gov NCT04762628, registered 21/02/2021, https://www. CLINICALTRIALS: gov/ct2/show/NCT04762628 .

The Exploitation of the Glycosylation Pattern in Asthma: How We Alter Ancestral Pathways to Develop New Treatments.

Asthma has reached epidemic levels, yet progress in developing specific therapies is slow. One of the main reasons for this is the fact that asthma is an umbrella term for various distinct subsets. Due to its high heterogeneity, it is difficult to establish biomarkers for each subset of asthma and to propose endotype-specific treatments. This review focuses on protein glycosylation as a process activated in asthma and ways to utilize it to develop novel biomarkers and treatments. We discuss known and relevant glycoproteins whose functions control disease development. The key role of glycoproteins in processes integral to asthma, such as inflammation, tissue remodeling, and repair, justifies our interest and research in the field of glycobiology. Altering the glycosylation states of proteins contributing to asthma can change the pathological processes that we previously failed to inhibit. Special emphasis is placed on chitotriosidase 1 (CHIT1), an enzyme capable of modifying LacNAc- and LacdiNAc-containing glycans. The expression and activity of CHIT1 are induced in human diseased lungs, and its pathological role has been demonstrated by both genetic and pharmacological approaches. We propose that studying the glycosylation pattern and enzymes involved in glycosylation in asthma can help in patient stratification and in developing personalized treatment.

The Cerebrospinal Fluid Free-Glycans Hex1 and HexNAc1Hex1Neu5Ac1 as Potential Biomarkers of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, affecting a growing number of elderly people. In order to improve the early and differential diagnosis of AD, better biomarkers are needed. Glycosylation is a protein post-translational modification that is modulated in the course of many diseases, including neurodegeneration. Aiming to improve AD diagnosis and differential diagnosis through glycan analytics methods, we report the glycoprotein glycome of cerebrospinal fluid (CSF) isolated from a total study cohort of 262 subjects. The study cohort consisted of patients with AD, healthy controls and patients suffering from other types of dementia. CSF free-glycans were also isolated and analyzed in this study, and the results reported for the first time the presence of 19 free glycans in this body fluid. The free-glycans consisted of complete or truncated N-/O-glycans as well as free monosaccharides. The free-glycans Hex1 and HexNAc1Hex1Neu5Ac1 were able to discriminate AD from controls and from patients suffering from other types of dementia. Regarding CSF N-glycosylation, high proportions of high-mannose, biantennary bisecting core-fucosylated N-glycans were found, whereby only about 20% of the N-glycans were sialylated. O-Glycans and free-glycan fragments were less sialylated in AD patients than in controls. To conclude, this comprehensive study revealed for the first time the biomarker potential of free glycans for the differential diagnosis of AD.

GlycA and CRP Are Genetically Correlated: Insight into the Genetic Architecture of Inflammageing.

Inflammageing is a condition of perpetual low-grade inflammation induced by ageing. Inflammageing may be predicted by the C-reactive protein (CRP) or by a recently described biomarker which measures N-glycosylated side chains of the carbohydrate component of several acute-phase proteins known as GlycA. The objective of this study was to examine in depth the genetic relationships between CRP and GlycA as well as between each of them and other selected cytokines, which may shed light on the mechanisms of inflammageing. Using the Olink 96 Inflammation panel, data on inflammatory mediators for 1518 twins from the TwinsUK dataset were acquired. Summary statistics for genome-wide association studies for several cytokines as well as CRP and GlycA were collected from public sources. Extensive genetic correlation analyses, colocalization and genetic enrichment analyses were carried out to detect the shared genetic architecture between GlycA and CRP. Mendelian randomization was carried out to assess potential causal relationships. GlycA predicted examined cytokines with a magnitude twice as great as that of CRP. GlycA and CRP were significantly genetically correlated (Rg = 0.4397 ± 0.0854, p-value = 2.60 × 10-7). No evidence of a causal relationship between GlycA and CRP, or between these two biomarkers and the cytokines assessed was obtained. However, the aforementioned relationships were explained well by horizontal pleiotropy. Five exonic genetic variants annotated to five genes explain the shared genetic architecture observed between GlycA and CRP: IL6R, GCKR, MLXIPL, SERPINA1, and MAP1A. GlycA and CRP possess a shared genetic architecture, but the relationship between them appears to be modest, which may imply the promotion of differing inflammatory pathways. GlycA appears to be a more robust predictor of cytokines compared to CRP.

Complexity and modification of the bull sperm glycocalyx during epididymal maturation.

Mammalian spermatozoa have a surface covered with glycocalyx, consisting of heterogeneous glycoproteins and glycolipids. This complexity arises from diverse monosaccharides, distinct linkages, various isomeric glycans, branching levels, and saccharide sequences. The glycocalyx is synthesized by spermatozoa developing in the testis, and its subsequent alterations during their transit through the epididymis are a critical process for the sperm acquisition of fertilizing ability. In this study, we performed detailed analysis of the glycocalyx on the sperm surface of bull spermatozoa in relation to individual parts of the epididymis using a wide range (24) of lectins with specific carbohydrate binding preferences. Fluorescence analysis of intact sperm isolated from the bull epididymides was complemented by Western blot detection of protein extracts from the sperm plasma membrane fractions. Our experimental results revealed predominant sequential modification of bull sperm glycans with N-acetyllactosamine (LacNAc), followed by subsequent sialylation and fucosylation in a highly specific manner. Additionally, variations in the lectin detection on the sperm surface may indicate the acquisition or release of glycans or glycoproteins. Our study is the first to provide a complex analysis of the bull sperm glycocalyx modification during epididymal maturation.

Sweet complexity: O-linked protein glycosylation in pathogenic Neisseria.

The genus Neisseria, which colonizes mucosal surfaces, includes both commensal and pathogenic species that are exclusive to humans. The two pathogenic Neisseria species are closely related but cause quite different diseases, meningococcal sepsis and meningitis (Neisseria meningitidis) and sexually transmitted gonorrhea (Neisseria gonorrhoeae). Although obvious differences in bacterial niches and mechanisms for transmission exists, pathogenic Neisseria have high levels of conservation at the levels of nucleotide sequences, gene content and synteny. Species of Neisseria express broad-spectrum O-linked protein glycosylation where the glycoproteins are largely transmembrane proteins or lipoproteins localized on the cell surface or in the periplasm. There are diverse functions among the identified glycoproteins, for example type IV biogenesis proteins, proteins involved in antimicrobial resistance, as well as surface proteins that have been suggested as vaccine candidates. The most abundant glycoprotein, PilE, is the major subunit of pili which are an important colonization factor. The glycans attached can vary extensively due to phase variation of protein glycosylation (pgl) genes and polymorphic pgl gene content. The exact roles of glycosylation in Neisseria remains to be determined, but increasing evidence suggests that glycan variability can be a strategy to evade the human immune system. In addition, pathogenic and commensal Neisseria appear to have significant glycosylation differences. Here, the current knowledge and implications of protein glycosylation genes, glycan diversity, glycoproteins and immunogenicity in pathogenic Neisseria are summarized and discussed.

Microfibril-associated glycoprotein 4 forms octamers that mediate interactions with elastogenic proteins and cells.

Microfibril-associated glycoprotein 4 (MFAP4) is a 36-kDa extracellular matrix glycoprotein with critical roles in organ fibrosis, chronic obstructive pulmonary disease, and cardiovascular disorders, including aortic aneurysms. MFAP4 multimerises and interacts with elastogenic proteins, including fibrillin-1 and tropoelastin, and with cells via integrins. Structural details of MFAP4 and its potential interfaces for these interactions are unknown. Here, we present a cryo-electron microscopy structure of human MFAP4. In the presence of calcium, MFAP4 assembles as an octamer, where two sets of homodimers constitute the top and bottom halves of each octamer. Each homodimer is linked together by an intermolecular disulphide bond. A C34S missense mutation prevents disulphide-bond formation between monomers but does not prevent octamer assembly. The atomic model, built into the 3.55 Å cryo-EM map, suggests that salt-bridge interactions mediate homodimer assembly, while non-polar residues form the interface between octamer halves. In the absence of calcium, an MFAP4 octamer dissociates into two tetramers. Binding studies with fibrillin-1, tropoelastin, LTBP4, and small fibulins show that MFAP4 has multiple surfaces for protein-protein interactions, most of which depend upon MFAP4 octamer assembly. The C34S mutation does not affect these protein interactions or cell interactions. MFAP4 assemblies with fibrillin-1 abrogate MFAP4 interactions with cells.

Defining albumin as a glycoprotein with multiple N-linked glycosylation sites.

BACKGROUND: Glycosylation is an enzyme-catalyzed post-translational modification that is distinct from glycation and is present on a majority of plasma proteins. N-glycosylation occurs on asparagine residues predominantly within canonical N-glycosylation motifs (Asn-X-Ser/Thr) although non-canonical N-glycosylation motifs Asn-X-Cys/Val have also been reported. Albumin is the most abundant protein in plasma whose glycation is well-studied in diabetes mellitus. However, albumin has long been considered a non-glycosylated protein due to absence of canonical motifs. Albumin contains two non-canonical N-glycosylation motifs, of which one was recently reported to be glycosylated. METHODS: We enriched abundant serum proteins to investigate their N-linked glycosylation followed by trypsin digestion and glycopeptide enrichment by size-exclusion or mixed-mode anion-exchange chromatography. Glycosylation at canonical as well as non-canonical sites was evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of enriched glycopeptides. Deglycosylation analysis was performed to confirm N-linked glycosylation at non-canonical sites. Albumin-derived glycopeptides were fragmented by MS3 to confirm attached glycans. Parallel reaction monitoring was carried out on twenty additional samples to validate these findings. Bovine and rabbit albumin-derived glycopeptides were similarly analyzed by LC-MS/MS. RESULTS: Human albumin is N-glycosylated at two non-canonical sites, Asn68 and Asn123. N-glycopeptides were detected at both sites bearing four complex sialylated glycans and validated by MS3-based fragmentation and deglycosylation studies. Targeted mass spectrometry confirmed glycosylation in twenty additional donor samples. Finally, the highly conserved Asn123 in bovine and rabbit serum albumin was also found to be glycosylated. CONCLUSIONS: Albumin is a glycoprotein with conserved N-linked glycosylation sites that could have potential clinical applications.

Lipidomic Comparisons of Whole Cream Buttermilk Whey and Cheese Whey Cream Buttermilk of Caprine Milk.

Buttermilk is a potential material for the production of a milk fat globule membrane (MFGM) and can be mainly classified into two types: whole cream buttermilk and cheese whey cream buttermilk (WCB). Due to the high casein micelle content of whole cream buttermilk, the removal of casein micelles to improve the purity of MFGM materials is always required. This study investigated the effects of rennet and acid coagulation on the lipid profile of buttermilk rennet-coagulated whey (BRW) and buttermilk acid-coagulated whey (BAW) and compared them with WCB. BRW has significantly higher phospholipids (PLs) and ganglioside contents than BAW and WCB. The abundance of arachidonic acid (ARA)- and eicosapentaenoic acid (EPA)-structured PLs was higher in WCB, while docosahexaenoic acid (DHA)-structured PLs were higher in BRW, indicating that BRW and WCB intake might have a greater effect on improving cardiovascular conditions and neurodevelopment. WCB and BRW had a higher abundance of plasmanyl PL and plasmalogen PL, respectively. Phosphatidylcholine (PC) (28:1), LPE (20:5), and PC (26:0) are characteristic lipids among BRW, BAW, and WCB, and they can be used to distinguish MFGM-enriched whey from different sources.

Galf-Specific Neolectins: Towards Promising Diagnostic Tools.

In the absence of naturally available galactofuranose-specific lectin, we report herein the bioengineering of GalfNeoLect, from the first cloned wild-type galactofuranosidase (Streptomyces sp. strain JHA19), which recognises and binds a single monosaccharide that is only related to nonmammalian species, usually pathogenic microorganisms. We kinetically characterised the GalfNeoLect to confirm attenuation of hydrolytic activity and used competitive inhibition assay, with close structural analogues of Galf, to show that it conserved interaction with its original substrate. We synthetised the bovine serum albumin-based neoglycoprotein (GalfNGP), carrying the multivalent Galf units, as a suitable ligand and high-avidity system for the recognition of GalfNeoLect which we successfully tested directly with the galactomannan spores of Aspergillus brasiliensis (ATCC 16404). Altogether, our results indicate that GalfNeoLect has the necessary versatility and plasticity to be used in both research and diagnostic lectin-based applications.