In‐depth comparison of N‐glycosylation of human plasma‐derived factor VIII and different recombinant products: from structure to clinical implications

Essentials

• Glycosylation heterogeneity of recombinant proteins affects pharmacokinetics and immunogenicity.
• N‐glycomics/glycoproteomics of plasma‐derived Factor VIII and 6 recombinant FVIIIs were compared.
• Depending on cell line, significant differences to plasma‐derived FVIII were observed.
• Recombinant FVIIIs expressed distinct and immunologically relevant epitopes.

Summary

Background/Objective

Human factor VIII (FVIII) is a plasma glycoprotein, defects of which result in hemophilia A. Current substitution therapy uses FVIII products purified from human plasma or from various cell lines (recombinant FVIII) with different levels of B‐domain deletion. Glycosylation is a post‐translational protein modification in FVIII that has a substantial influence on its physical, functional and antigenic properties. Variation in glycosylation is likely to be the reason that FVIII products differ in their pharmacokinetics, pharmacodynamics and immunogenicity. However, the literature on FVIII glycosylation is inconsistent, preventing assembly into a coherent model. Seeking to better understand the glycosylation mechanisms underlying FVIII biology, we studied the N‐glycosylation of human plasma‐derived (pd)FVIII and six rFVIII products expressed in CHO, BHK or HEK cell lines.

Methods

FVIII samples were subjected to head‐to‐head detailed glycomic and glycoproteomic characterization using a combination of MALDI‐MS and MS/MS, GC‐MS and UPLC‐UV‐MS^E technologies.

Results/Conclusion

The results of our study detail the N‐glycan repertoire of pdFVIII to an unprecedented level, and for the first time, provide evidence of N‐glycolylneuraminic acid (NeuGc) found on pdFVIII. Although site‐specific glycosylation of rFVIII proved consistent with pdFVIII regardless of the expression system, the entire N‐glycan content of each sample appeared significantly different. Although the proportion of biologically important epitopes common to all samples (i.e. sialylation and high‐mannose) varied between samples, some recombinant products expressed distinct and immunologically relevant epitopes, such as LacdiNAc (LDN), fucosylated LacdiNAc (FucLDN), NeuGc, Lewis^X/Y and Gal_α1,3Gal epitopes. rFVIII expressed in HEK cells showed the greatest glycomic differences to human pdFVIII.     

α-突触核蛋白与Cu~(2+)相互作用的研究进展

帕金森病是一种慢性中枢神经系统退行性疾病,其主要的病理特征是黑质致密部多巴胺能神经元丢失,残存神经元胞质中出现路易小体,其主要成分是异常聚集的α-synuclein蛋白(α-突触核蛋白)。α-synuclein的聚集与多种因素有关,其中金属离子与α-synuclein结合可导致蛋白构象发生变化引起蛋白发生聚集。近年的研究发现,Cu2+能够特异地结合α-synuclein蛋白,并诱导α-synuclein的聚集。该文就Cu2+与α-synuclein相互作用的的结合位点、结合模式以及Cu2+在α-synuclein毒性形式中可能发挥的作用作一综述。     

Cryptic deletions and inversions of chromosome 21 in a phenotypically normal infant with transient abnormal myelopoiesis: a molecular cytogenetic study

A case of transient abnormal myelopoiesis in a normal newborn without features of Down syndrome is described. The majority of bone marrow cells analysed belonged to a chromosomally abnormal clone with trisomy for chromosomes 18 and 21. Complex intrachromosomal rearrangements of one chromosome 21, demonstrated by fluorescence in situ hybridization using locus-specific probes, were found in a minor population of the clonal cells. These rearrangements involved loci previously shown to be rearranged in the leukaemic cells from patients with Down syndrome and leukaemia. However, the child's myeloproliferation resolved rapidly, with disappearance of the abnormal clone, and 3.5 years later she remains well.     

晚期糖化终末产物在去卵巢大鼠骨质疏松发病中的作用及氨基胍防治效果

目的　探讨晚期糖化终末产物 (AGEs)在去卵巢大鼠骨质疏松发病中的作用及氨基胍防治效果。方法　选用 1 0月龄大鼠通过卵巢切除诱导骨质疏松并用氨基胍防治其骨量丢失 ,测定其骨密度及骨胶原中 AGEs的含量及血、尿生化指标。结果　卵巢切除大鼠骨密度 (0 .2 0 7g/cm2 ± 0 .0 1 6 g/cm2 )明显低于假手术大鼠 (0 .2 36 g/cm2 ± 0 .0 1 0 g/cm2 ) (P<0 .0 1 ) ,而骨胶原 AGEs含量 (59.86 RU/mg胶原± 3.1 3 RU/mg胶原 )明显高于假手术组大鼠 (53.83RU/mg胶原± 5.46RU/mg胶原 ) (P<0 .0 1 )。卵巢切除大鼠血清雌激素 (4.50 pg/ml± 1 .73pg/ml)与假手术组(8.45 pg/ml± 2 .90 pg/ml)比较明显降低 (P<0 .0 2 ) ,2 4 h尿钙、尿钙与肌酐比值、尿磷、尿磷与肌酐比值均有升高趋势。卵巢切除大鼠给予氨基胍后骨密度 (0 .2 2 1 g/cm2± 0 .0 1 7g/cm2 )明显升高 (P<0 .0 5) ,骨胶原中 AGEs含量 (51 .2 3RU/mg胶原± 7.46RU/mg胶原 )明显降低 (P<0 .0 1 ) ,血清雌激素 (8.59pg/ml± 3.0 1 pg/ml)明显升高 (P<0 .0 1 ) ,2 4 h尿钙、尿钙与肌酐比值降低 ,2 4 h尿羟脯氨酸升高 ,与手术组比较有显著差异 (P<0 .0 5)。结论　卵巢切除后 ,雌激素的降低和 AGEs的增加是导致骨质疏松的主要原因。而氨基胍通过降低体内 AGE     

Glycosylation enhances oxygen radical-induced modifications and decreases acetylhydrolase activity of human low density lipoprotein

Background. Posttranslational nonenzymatic glycosylation of native low-density lipoprotein (n-LDL) occurs bothin vitro andin vivo in diabetic patients. Glycosylated LDL (glc-LDL) behave similarly to oxidized LDL in some respects. In fact, unlike n-LDL, uptake of glc-LDL can occur in part by the scavenger receptor(s), as also demonstrated for oxidized LDL. The enzyme acetylhydrolase, carried by LDL, catabolizes platelet activating factor (PAF). This enzymatic activity is inhibited in oxidized LDL. However, it is unknown whether glc-LDL have reduced acetylhydrolase activity.Objectives. The first aim of the study was to investigate whether glc-LDL were more susceptible than n-LDL to oxidative modification, and which different oxygen radical species were involved in the phenomenon. Moreover, in order to investigate whether glycosylation may affect acetylhydrolase, we also measured this enzymatic activity in both n- and glc-LDL.Methods. In vitro glc-LDL and n-LDL were exposed to the oxidants xanthine/xanthine oxidase (X/XO; 2 mM and 100 mU/ml, respectively), or CuSO₄ (10 M) for 18 hs at 37°C. Parallel experiments were done in the presence of the superoxide radical scavenger superoxide dismutase (SOD; 330 U/ml), the hydrogen peroxide scavenger catalase (1000 U/ml), or the hydroxyl radical scavenger dimethylthiourea (10 mM) or dimethylsulfoxide (1 mM). Standards of PAF and lyso-PAF were visualized with iodine vapors after separation by thin layer chromatography. The distribution of label was determined by an imaging scanner. Labeled products were then isolated from the chromatography plate, and the amount of³H-lyso-PAF formed was determined by liquid scintillation counting.Results. Glc-LDL were more susceptible than n-LDL to lipid peroxidation (n-LDL 22.9±3.4 vs 34.8±4.2^* nmoles/MDA/mg of protein in glc-LDL oxidized by X/XO and n-LDL 28.9±4.2 vs 40.4±4.1^* in glc-LDL oxidized by CuSO₄,^*p<0.05 vs n-LDL). SOD, but not other scavengers, prevented peroxidation, indicating an obligatory role for superoxide radicals. Oxidation of glc-LDL also induced a higher degree of apolipoprotein-B₁₀₀ modifications than n-LDL, with increased electrophoresis mobility and decreased TNBS reactivity. These effects were similarly prevented by SOD. Finally, acetylhydrolase activity was significantly lower in glc-LDL than in n-LDL.Conclusion. Glycosylation increases LDL oxidation due to superoxide radicals, and also reduces acetylhydrolase activity. These phenomenona may contribute to enhance and/or accelerate the progression of atherosclerosis in diabetic patients.Abbreviation LDL low density lipoprotein - n-LDL native LDL - glc-LDL glycosylated LDL - PAF platelet activating factor - X/XO xanthine/xanthine oxidase reaction - SOD superoxide dismutase - DMTU dimethylthiourea - DMSO dimethylsulfoxide - TNBS trinitrobenzenesulfonic acid - MDA malonyldialdehyde - LPO lipid peroxides This study was presented in abstract form at the 42nd Annual Scientific Session of the American College of Cardiology, Anaheim, CA, 14–18 March 1993 (see Ref. 30).     

Congenital disorders of glycosylation type IIb with MOGS mutations cause early infantile epileptic encephalopathy,dysmorphic features,and hepatic dysfunction

AimMOGS mutations cause congenital disorders of glycosylation type IIb (CDG-IIb or GCS1-CDG). The specific manifestations caused by the mutations in this gene remain unknown. We aimed to describe the clinical features of CDG- IIb and the effectiveness of urinary oligosaccharide analysis in the diagnosis of CDG- IIb.MethodsPatient 1 was analyzed with whole-exome sequencing (WES) to identify the causative gene of intractable epilepsy and severe developmental delay. After detecting MOGS mutation in patient 1, we analyzed patients 2 and 3 who were siblings and had clinical features similar to those in patient 1. Urinary oligosaccharide analysis was performed to confirm CDG- IIb diagnosis in patient 1. The clinical features of these patients were analyzed and compared with those in eight published cases.ResultsOur three patients presented with early infantile epileptic encephalopathy, generalized hypotonia, hepatic dysfunction and dysmorphic features. In two cases, compound heterozygous mutations in MOGS were identified by WES. Isolation and characterization of the urinary oligosaccharide was performed in one of these cases to confirm the diagnosis of CDG-IIb. Although the isoelectric focusing of transferrin (IEF-T) of serum in this patient was normal, urinary excretion of Hex₄ corresponding to Glc₃Man was observed by mass spectrometry.ConclusionThis report provides clinical manifestations of CDG-IIb with MOGS mutation. CDG-IIb shows a normal IEF profile of serum transferrin and cannot be detected by structural analysis of the patient’s glycoproteins. Characterization of urinary oligosaccharides should be considered to detect this disorder.     

Novel ALG12 variants and hydronephrosis in siblings with impaired N-glycosylation

BackgroundALG12-CDG is a rare autosomal recessive type I congenital disorder of glycosylation (CDG) due to pathogenic variants in ALG12 which encodes the dolichyl-P-mannose:Man-7-GlcNAc-2-PP-dolichyl-alpha-6-mannosyltransferase. Thirteen patients from unrelated 11 families have been reported, most of them result in broad multisystem manifestations with clinical variability. It is important to validate abnormal glycosylation to establish causal relationship.Case reportHere, we report two siblings with novel compound heterozygous variants in ALG12: c.443T>C, p.(Leu148Pro) and c.412_413insCGT, p.(Gln137_Phe138insSer). Both patients showed global developmental delay, microcephaly, hypotonia, failure to thrive, facial dysmorphism, skeletal malformations and coagulation abnormalities, which are common in ALG12-CDG. In addition, one of our patients showed left hydronephrosis, which is a novel clinical feature in ALG12-CDG. Brain MRI showed hypoplasia of cerebrum, brain stem and cerebellar vermis in both patients. N-glycosylation defects of trypsin digested transferrin peptides were revealed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and electrospray ionization MS verified the lack of N-glycans in transferrin.ConclusionsThe present study can add hydronephrosis to phenotypic spectrum of ALG12-CDG. Since the symptoms of ALG12-CDG are quite diverse, the combination of whole-exome sequencing and transferrin glycopeptide analysis with MS, can help diagnosis of ALG12-CDG.     

0.5 Mb Array as a First‐Line Prenatal Cytogenetic Test in Cases without Ultrasound Abnormalities and Its Implementation in Clinical Practice

Using whole‐genome array testing instead of karyotyping in prenatal diagnosis for all indications may be desirable because of the higher diagnostic yield and shorter reporting time. The goal of this research was finding the optimal array resolution that could replace routine prenatal karyotyping in cases without ultrasound abnormalities, for example, referred for advanced maternal age or abnormal first trimester screening. As variants of unknown clinical significance (VOUS), if reported, might complicate decision‐making about continuation of pregnancy, such an optimal array resolution should have a high abnormality detection rate and reveal a minimal amount of VOUS. The array data of 465 fetuses were retrospectively evaluated with several resolution levels, and the Decipher microdeletion/microduplication syndrome list was reviewed to assess what could be theoretically missed with a lower resolution. A 0.5‐Mb resolution showed a high diagnostic yield potential and significantly minimized the number of VOUS. Based on our experience, we recommend genomic SNP array as a first‐tier test in prenatal diagnosis. The resolution should be chosen based on the indication. In cases of fetal ultrasound abnormalities or intrauterine fetal death (IUFD), high‐resolution analysis should be done. In other cases, we advise replacing karyotyping by SNP array analysis with 0.5 Mb resolution.     

Skin Advanced Glycation End Product Accumulation Is Poorly Reflected by Glycemic Control in Type 2 Diabetic Patients (ZODIAC-9)

Background

Glycemic memory can be reflected by tissue accumulation of advanced glycation end products (AGEs). In type 1 diabetes mellitus (T1DM) patients, hemoglobin A1c (HbA1c) levels over various time periods poorly predicted the accumulation of different AGEs in skin biopsies. Our aim was to investigate whether HbA1c assessments can predict the change in skin AGEs during time in type 2 diabetes mellitus (T2DM).

Methods

We included 452 T2DM patients participating in a shared-care setting, who are screened annually for HbA1c and diabetic complications. Baseline and follow-up levels of skin AGEs were assessed with a validated noninvasive autofluorescence (AF) method, which is based on the fluorescence characteristics of certain AGEs.

Results

Our study population had a mean age of 65 years and 54% were female. After a mean follow-up duration of 3.3 years, linear regression analyses showed weak relationships among different assessments of HbA1c (baseline, maximum, mean, and variance of HbA1c) and skin AF at follow-up. Baseline skin AF and age were predictors of skin AF at follow-up, but diabetes duration, smoking, and creatinine were of less or no predictive value for skin AF at follow-up.

Conclusions

In our T2DM population, integrated HbA1c assessments over years poorly predict the change in skin AGE level measured by skin AF. These findings agree with results in patients with T1DM. This suggests either the need for longer exposure to glucose disturbances to change tissue AGEs or other mechanisms, such as oxidative stress, leading to AGE accumulation.