key: cord-0964713-2kyls70p
authors: Friganović, Tomislav; Tomašić, Antonela; Šeba, Tino; Biruš, Ivan; Kerep, Robert; Borko, Valentina; Šakić, Davor; Gabričević, Mario; Weitner, Tin
title: Low-pressure chromatographic separation and UV/Vis spectrophotometric characterization of the native and desialylated human apo-transferrin
date: 2021-09-20
journal: Heliyon
DOI: 10.1016/j.heliyon.2021.e08030
sha: ea72735e0443050ada4b4513e044e05b60b3599b
doc_id: 964713
cord_uid: 2kyls70p

Low-pressure pH gradient ion exchange separation provides a fast, simple and cost-effective method for preparative purification of native and desialylated apo-transferrin. The method enables easy monitoring of the extent of the desialylation reaction and also the efficient separation and purification of protein fractions after desialylation. The N-glycan analysis shows that the modified desialylation protocol successfully reduces the content of the sialylated fractions relative to the native apo-transferrin. In the optimized protocol, the desialylation capacity is increased by 150 %, compared to the original protocol provided by the manufacturer. The molar absorption coefficients in the near-UV region for the native and desialylated apo-transferrin differ by several percent, suggesting a subtle dependence of the glycoprotein absorbance on the variable sialic acid content. The method can easily be modified for other glycoproteins and is particularly appropriate for quick testing of sialic acid content in the protein glycosylation patterns prior to further verification by mass spectrometry.

Glycosylation is one of the most common posttranslational modifications of proteins. Nearly all membrane and secreted proteins, as well as numerous intracellular proteins, are modified with complex glycan structures to enable communication, binding, recognition and/or modification of the protein activity. Such modified proteins play a role in almost every biological process and are involved in numerous major diseases [1] . Glycan moieties of glycoproteins are not synthesized using a direct genetic template. Instead, they result from the interplay of several hundred enzymes organized in complex pathways. Increase of interest for glycosylation and other associated processes resulted in the opening of a new field in biology named glycobiology [2, 3] . Changes in the glycosylation pattern can have an important role in cellular recognition and the regulation of gene expression, in addition to the influence on function of proteins. Furthermore, a change of the glycosylation pattern has been associated with numerous pathological conditions [4] .

Transferrin is a heavily glycosylated serum protein that binds to and consequently mediates the cellular transport of iron. Reference range of the human serum levels is 1940-3420 mg/L, but this may be increased during pregnancy, therapy with oral contraceptives and/or due to increased synthesis caused by iron deficiency. Lower values are characteristic for increased catabolism, liver problems, chronic infections, malnutrition, trauma etc. Half-life of transferrin in the serum is about 16 hours. [5] . Transferrin structure consists of 679 amino acids with two glycan structures covalently linked to asparagine residues 413 and 611. Glycan structures can be bi-or tri-antennary and each of them terminates with sialic acid. In normal serum, 85 % is tetra-sialotransferrin and the rest (15 %) is penta-or tri-sialotransferrin [6] . The scheme of human transferrin glycoforms microheterogeneity is shown in Figure 1 . Gene mutations can cause defects in glycosylation resulting in inborn errors of metabolism, characterized by deficient or reduced glycosylation and known as congenital disorders of glycosylation (CDGs) [7] . Change of sialylation has also been linked to alcoholism and many pathological states [8, 9] . Nowadays, the analysis of glycosylation change is used as a diagnostic tool for alcoholism and congenital disorders of glycosylation [10, 11] . Importantly, the sialylation of transferrin may alter its fundamental function as iron carrier and may also affect the transfer of iron into liver [12, 13] .

Ferroptosis, a newly identified form of non-apoptotic regulated cell death characterized by iron-dependent accumulation of lipid peroxides plays a vital role in the treatment of tumours, renal failure or ischemia reperfusion injury [14] . Both transferrin and transferrin receptor 1 (TFR1) are required for ferroptosis induction [15] , and this might provide new implications for the function of transferrin sialylation patterns. Recently, it has been hypothesized that ferroptosis may be an important cause of multiple organ involvement in severe coronavirus disease 2019 (COVID-19) for a substantial proportion of patients who have lymphopenia, low serum iron levels, and multiple organ involvement [16] . Severe COVID-19 disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has also been associated with disseminated intravascular coagulation and thrombosis, accompanied by an upregulated expression of transferrin in SARS-CoV-2-infected cells [17] . Notably, transferrin has been identified an important clotting regulator and an adjuster in the maintenance of blood coagulation balance [18] . Another recent report indicates that transferrin receptor is possible entry point for SARS-CoV-2 and a promising anti-COVID-19 target [19] .

For further mechanistic studies, it is crucial to develop robust methods of preparation and characterization of transferrin sialoforms. The purpose of this study is to define the optimal transferrin desialylation procedure and then separate the desialylated apo-transferrin (Tf-S) from the native apo-transferrin (TfþS) using low-pressure pH gradient ion exchange chromatography. Detailed N-glycan analysis and UV/Vis spectrophotometric characterization of the obtained Tf-S and TfþS fractions is provided as a first step towards detailed iron binding and/or release studies.

Native human apo-transferrin (Biorbyt, UK, cat. no. orb80927), sodium acetate trihydrate (Kemika, Croatia), calcium chloride (Lach-Ner, Croatia), sodium chloride (Kemika, Croatia), neuraminidase (Glyco-Cleave® Neuraminidase Kit, GALAB Technologies, Germany, cat. no. 132011), pISep Buffer Kit (CryoBioPhysica, USA, cat. no. 20055), hydrogen chloride (Carlo Erba Reagents, Italy, 37 % solution), sodium hydroxide (Kemika, Croatia, pellets 2-5 mm), sodium phosphate (Kemika, Croatia), guanidine hydrochloride (PanReac AppliChem, USA), MES (2-(N-morpholino)ethanesulfonic acid, Sigma Aldrich, USA) and potassium chloride (Alkaloid, North Macedonia) were used without further purification. Water used for experiments was double distilled in an all-glass apparatus. All experiments except the enzymatic desialylation were performed at room temperature.

Desialylated apo-transferrin is prepared by incubation of immobilized neuraminidase enzyme beads suspension (Glycocleave) in the native apotransferrin buffered stock solution (pH ¼ 5.5, t ¼ 38 C). After the incubation period of 48 hours, the desialylated sample is collected, washed out and concentrated by centrifugal filtration. The complete protocol has been described elsewhere [20] .

Sialoform separation is performed by using specialized pH gradient ion exchange chromatography buffers (pIsep). The mixture of fully desialylated apo-transferrin and native apo-transferrin is dissolved in the start buffer pIsep A (pH ¼ 8) and injected onto HiTrap Q HP anion exchange chromatography columns (Cytiva, USA). Two 1 mL columns were serially connected for improved separation. Elution is done by single step linear gradient (0-100 % pIsep B, pH ¼ 4) procedure using € AKTA Start Table 1 . Structure and content of N-glycans in the native and desialylated apo-transferrin, TfþS and Tf-S, respectively, as determined by UPLC-MS. The N-glycan composition was determined by MS and the percent content of individual structures was calculated from the integrals of corresponding UPLC fluorescence signals [24] . The dominant fractions with a content !5% are printed in bold and make up approximately 90% of the total protein. The IS value corresponds to the proposed index of sialylation defined in Eq. (1).

protein purification system (Cytiva, USA). Protein concentration in the eluate is monitored by measuring absorbance at λ ¼ 280 nm and protein fraction recovery can be calculated by integration over surface area (mL Â mAU). After separation, the pH value of each fraction containing eluted protein was measured, corresponding to the approximate protein isoelectric point, pI. Full details of the pH-gradient chromatography have been described elsewhere [21] .

In order to verify the results of the enzymatic desialylation of the native protein and pH-gradient separation of different sialoforms, the complete N-glycan profiling of TfþS and Tf-S was performed. Briefly, the protein N-glycans were released with the addition of 1.2 U of PNGase F (Promega, USA) and overnight incubation at 37 C. The released N-glycans were labeled with 2-aminobenzamide (Sigma Aldrich, USA) and purified using hydrophilic interaction liquid chromatography solid-phase extraction (HILIC-SPE). Fluorescently labeled N-glycans were separated by Acquity UPLC H-Class instrument (Waters, USA) using BEH Glycan chromatography column (Waters, USA). All glycan structures were annotated with MS/MS analysis using Synapt G2-Si ESI-QTOF-MS system (Waters, USA). Glycan compositions and structural features were assigned using software tools GlycoWorkbench and Glycomode, according to obtained MS and MS/MS spectra [22, 23] . Full details of the protein characterization by UPLC-MS have been described elsewhere [24] .

In order to facilitate the determination of protein quantities in mechanistic studies, the molar absorption coefficients of both the native and desialylated protein were determined according to the modified Edelhoch method, as described elsewhere [25] . Briefly, the folded protein absorbance at 280 nm (A 280 ) was measured in 25 mM sodium phosphate buffer (pH ¼ 7.4). The unfolded (denatured) protein absorbance at 280 nm (A u 280 ) was measured in the same buffer in the presence of 6 M guanidine HCl. The molar absorption coefficient of a folded protein at 280 nm (ε 280 ) is then equal to the product of a reference molar absorption coefficient for the unfolded protein, ε u 280 , and the ratio of folded and unfolded protein absorbance, i.e. ε 280 ¼ ε u 280 Â A 280 /A u 280 . The reference value of ε u 280 ¼ 81080 has been calculated from the contributions of 8 tryptophan, 26 tyrosine and 19 cystine residues in apo-transferrin structure [26, 27, 28] . The UV/Vis measurements were [24] . Schematic N-glycan structures and the corresponding fluorescence signals are indicated by arrows: N-acetylglucosamine (blue), mannose (green), galactose (yellow), fucose (red), sialic acid (pink). Table 2 . Structure and content of N-glycans in the desialylated apo-transferrin, Tf-S, for two separate batches run at different times from the same original batch of the native protein, as determined by UPLC-MS. The N-glycan composition was determined as described in Table 1 All samples were prepared in triplicate and A 280 was measured for each sample in a quartz cell (l ¼ 1 cm) using Varian Cary 50 spectrophotometer (Varian, Australia). The data were analyzed using a single factor ANOVA routine in Microsoft Excel Data Analysis Toolpak. If the calculated P-value is more than the chosen confidence level (α ¼ 0.05), and the obtained F-value is less than the critical F-value, the nullhypothesis that there is no significant difference between the means of the samples should not be rejected [29] .

Compared to the original protocol [30] , the concentration of working buffer was increased from 0.05 M to 0.2 M, thus increasing the desialylation capacity from 2 mg of protein to 5 mg of protein per reaction cycle, corresponding to 150 % increase in the reaction throughput. The final ratio is 1 mL of the immobilized enzyme suspension per 25 mg of protein.

The desialylation enzyme is stable and can be used multiple times if an appropriate rinsing and preserving procedure is applied. However, the immobilized enzyme activity decreases after repeated use and increased incubation time is necessary to obtain comparable degree of protein desialylation. For successful desialylation, it is crucial to closely monitor the pH of the solution before and during the reaction (optimal pH ¼ 5.5) and adjust accordingly by the addition of alkali. The reaction releases terminal sialic acids and unchecked acidification of the reaction mixture can inactivate the enzyme.

Initial attempts to form of externally controlled pH gradient in the range from pH ¼ 8 to pH ¼ 4 using either Servalyt (SERVA Electrophoresis, Germany) or Pharmalyte (Cytiva, USA) buffers were unsuccessful, AE 0.2) Â 10 3 M À1 cm À1 ; Bottom: The difference in molar absorption coefficients, Δε, for the intact and denatured proteins: TfþS (black trace) and Tf-S (red trace). The values were calculated as Δε ¼ ε f -ε u , where ε f is the molar absorption coefficient of the intact (folded) protein, and ε u is the molar absorption coefficient of the denatured (folded) protein in 6 M guanidine [25] .

presumably due to insufficient buffering capacity at specific pH values (data not shown). However, a very linear pH gradient over the required pH range was achieved using pISep buffers that are specifically designed for chromatofocusing [31] . The pH gradient shown in Figure 2 (red trace) is linear in the range of 11-30 mL, corresponding to the range 7.78 > pH > 4.22 (R2 ¼ 0.999). An additional improvement achieved using pISep buffer was in a significantly reduced absorbance baseline at 280 nm, making it easier to monitor protein elution from the column and more accurately calculate the amount of eluted protein by integrating chromatograms. However, the precise preparation of chromatofocusing buffers requires the special pISep pH gradient maker software.

Further improvement in transferrin sialoform separation was achieved by connecting two 1 mL HiTrap Q HP columns in a series, as compared to using only one 1 mL column. The two-column series backpressure of 0.15 MPa was well within the operational range of the € AKTA Start system. The observed pI values for the native (pI % 5) and desialylated (pI % 6) apo-transferrin differ significantly and hence can be fully separated (Figure 2 ).

In order to confirm the results of the pH chromatofocusing, both elution fractions, TfþS and Tf-S, were analyzed by mass spectroscopy [24, 32] . The detailed N-glycan structure and quantification of transferrin sialoforms determined by UPLC-MS (Table 1 and Figure 3) shows that the native apo-transferrin (TfþS) is dominated by glycan structures with 1 or 2 terminal sialic acid (A2G2S1 and A2G2S2) which together make up approximately 90 % of the total glycan content. Conversely, the N-glycan structures without the terminal sialic acids (A2G2, FA2G2) are dominant in desialylated apo-transferrin (Tf-S) and together make up approximately 90 % of the total glycan content. The applied protocol requires 100 μg of protein and should be repeated each time a new commercial sample is purchased and also after each desialylation cycle.

The reproducibility of the N-glycan content was tested for two separate batches run at different times from the same original batch of the native protein. The first batch of Tf-S was prepared with the fresh enzyme, whereas the second batch of Tf-S was prepared with the enzyme that has been recycled a number of times. Due to the decreased activity of the recycled enzyme, the incubation time was increased from 2 days (for the fresh enzyme) to 9 days (for the recycled enzyme). However, the content differences are within 1 % for every N-glycan fraction, as shown in Table 2 .

According to the manufacturer's specifications, the used enzyme preferentially hydrolyzes α2,3 linkages of sialic acid, but will also cleave α2,6 and α2,8 linkages, with the preference for α2,3 linkages estimated at 260-fold [30] . This preference for α2,3 linked sialic acids might account for the observed incomplete desialylation of the native protein (approximately 10 % remaining sialylated N-glycan fractions). For the purpose of simple comparison of the overall protein sialic acid content for different samples we propose a simple measure, index of sialylation, defined in Eq. (1): [29] .

where IS is the index of sialylation, n is the number of the N-glycan fraction, f i is the % content of the particular N-glycan fraction and s i is the number of sialic acids in the structure of the same N-glycan fraction. The composition of the native protein sample in Table 1 yields the value IS (TfþS) ¼ 158.87. For comparison, the desialylated protein sample in Table 1 yields the value IS (Tf-S) ¼ 9.76, signifying 93.9 % reduction in the protein sialic acid content. Similar comparison of the desialylated protein samples in Table 2 yields only 0.58 % difference in the overall sialic acid content between Tf-S batches. Alternatively, this effect can also arise from small conformational change in the protein due to different solvation of TfþS and Tf-S caused by different surface charge. Such a small conformational change might indeed be the reason for the subtle variations in the TfþS and Tf-S difference spectra obtained by denaturation in 6 M guanidine (Figure 4 , Bottom) [25, 33, 34] . The measured absorbance of TfþS at 280 nm shows no significant dependence on salt concentration up to 1 M KCl or pH in the range 4.9 < pH < 7.6 ( Figure 5 ). The resulting P-value calculated using a single factor ANOVA routine in Microsoft Excel was greater than the default confidence level (P > 0.05), resulting in the acceptance of the null-hypothesis of equal means (Tables 3 and 4 ) [29] .

These results will allow the precise determination of the molar absorption coefficients of iron-saturated transferrin species, as well as the determination of the fluorescence properties of the proteins. The applied protocol requires 1 mg of protein, of which 60 % can be reused for other measurements. Similarly to the N-glycan analysis, the molar absorption coefficients determination should also be repeated for each new native protein batch and each desialylation cycle due to possible variable sialic acid content.

Low-pressure pH gradient ion exchange separation provides a fast, simple and cost-effective method for preparative purification of native and desialylated apo-transferrin. The method enables easy monitoring of the extent of the desialylation reaction and also the efficient separation and purification of protein fractions after the desialylation reaction is terminated. Furthermore, the method can easily be modified for other glycoproteins and is particularly appropriate for quick testing of protein sialic acid content prior to verification by mass spectrometry. The Nglycan analysis shows that the modified desialylation protocol successfully reduces the content of the sialylated fractions relative to the native apo-transferrin. In the optimized protocol, the desialylation capacity is increased by 150 %, compared to the original protocol provided by the manufacturer.

In order to ensure the reproducibility of any further mechanistic studies, the complete N-glycan assignation and molar coefficients determination should be performed for each new native protein batch, as well as after every desialylation cycle. This is important because different native protein batches might have different N-glycan profiles, depending on the protein source. Additionally, the decreased enzyme activity after repeated use requires extended incubation time for sufficient desialylation. Importantly, the molar absorption coefficients of the native and desialylated apo-transferrin differ by several percent, suggesting that the literature data on glycoprotein molar absorption coefficients should be taken with caution because the measurement depends on the N-glycan composition of the protein, which is variable. Davor Saki c: Analyzed and interpreted the data; Wrote the paper. Mario Gabri cevi c: Contributed reagents, materials, analysis tools or data; Analyzed and interpreted the data.

Tin Weitner: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper. 

Data will be made available on request.

The authors declare no conflict of interest.

No additional information is available for this paper. Table 3 .

Vertebrate protein glycosylation: diversity, synthesis and function

Essentials of Glycobiology

Searching for medicine's sweet spot

A retrospective and prospective view of glycopathology

Carbohydrate-deficient transferrin in serum: a new marker of potentially harmful alcohol consumption reviewed

Microheterogeneity of human serum transferrin: a biological phenomenon studied by isoelectric focusing in immobilized pH gradients

Insights into complexity of congenital disorders of glycosylation

Changes of carbohydrate-deficient transferrin in chronic, Alcoholism

Carbohydrate-deficient transferrin as a marker of chronic alcohol abuse: a critical review of preanalysis, analysis, and interpretation

Carbohydrate-deficient transferrin-a valid marker of alcoholism in population studies? Results from the Copenhagen City Heart Study

Prenatal diagnosis of congenital disorder of glycosylation type Ia (CDG-Ia) by cordocentesis and transferrin isoelectric focussing of serum of a 27-week fetus with non-immune hydrops

Rapid alterations in transferrin sialylation during sepsis

The behavior of asialotransferrin-iron in the rat

Mechanisms of ferroptosis and relations with regulated cell death: a review

Molecular mechanisms of ferroptosis and its role in cancer therapy

SARS-CoV-2 infection: can ferroptosis be a potential treatment target for multiple organ involvement?

COVID-19-Related coagulopathy-is transferrin a missing link

Transferrin plays a central role in coagulation balance by interacting with clotting factors

Transferrin receptor is another receptor for SARS-CoV-2 entry

Protocol for Enzymatic Desialylation of Native Apo-Transferrin, Zenodo

Protocol for pH-Gradient Chromatofocusing of the Native and Desialylated Human Apo-Transferrin, Zenodo

GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans

GlycoMod-a software tool for determining glycosylation compositions from mass spectrometric data

Protocol for Ultra Performance Liquid Chromatography-Mass Spectrometry N-Glycan Analysis of the Native and Desialylated Human Apo-Transferrin, Zenodo

Protocol for Spectrophotometric Determination of Native and Desialylated Apo-Transferrin Molar Absorption Coefficients, Zenodo

Spectrophotometric determination of protein concentration

Protocol to determine accurate absorption coefficients for iron-containing transferrins

Calculation of protein extinction coefficients from amino acid sequence data

Excel Scientific and Engineering Cookbook

Product Description for GlycoCleave® Neuraminidase Kit

Theory and applications of a novel ion exchange chromatographic technology using controlled pH gradients for separating proteins on anionic and cationic stationary phases

Association of Nglycosylation with breast carcinoma and systemic features using high-resolution quantitative UPLC

Ultraviolet Spectroscopy of Proteins

Biological macromolecules: UV-visible spectrophotometry

The N-glycan profiling by UPLC-MS was provided by the Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, Ante Kova ci ca 1, 10000 Zagreb, Croatia.