Recombinant Human NEU1, His-tagged

• Cat.No.: NEU1-156H
• Product Overview: Recombinant Human Sialidase-1 is produced by our mammalian expression system in human cells. The target protein is expressed with sequence (Glu48-Leu415) of Human NEU1 fused with a 6His tag at the C-terminus.

Specification

• Species: Human
• Source: HEK293
• Tag: His
• Description: Sialidase-1 belongs to the N-acetyl-a neuraminidase family. Sialidase-1 is expressed in many tissues; it is highly expressed in the pancreas, and weakly expressed in the brain. Sialidase-1 is a lysosomal enzyme, which cleaves terminal sialic acid residues from substrates such as glycoproteins and glycolipids. Deficiencies in the human enzyme Sialidase-1 leads to sialidosis, a rare lysosomal storage disease. Sialidase-1 has been shown to interact with Cathepsin A (protective protein), β-galactosidase and N-acetylgalactosamine-6-sulfate sulfatase in a multienzyme complex.
• AA Sequence: ENDFGLVQPLVTMEQLLWVSGRQIGSVDTFRIPLITATPRGTLLAFAEARKMSSSDEGAKFIALR RSMDQGSTWSPTAFIVNDGDVPDGLNLGAVVSDVETGVVFLFYSLCAHKAGCQVASTMLVWSKDD GVSWSTPRNLSLDIGTEVFAPGPGSGIQKQREPRKGRLIVCGHGTLERDGVFCLLSDDHGASWRY GSGVSGIPYGQPKQENDFNPDECQPYELPDGSVVINARNQNNYHCHCRIVLRSYDACDTLRPRDV TFDPELVDPVVAAGAVVTSSGIVFFSNPAHPEFRVNLTLRWSFSNGTSWRKETVQLWPGPSGYSS LATLEGSMDGEEQAPQLYVLYEKGRNHYTESISVAKISVYGTLVDHHHHHH
• Endotoxin: Less than 0.1 ng/μg (1 IEU/μg).
• Purity: Greater than 95% as determined by reducing SDS-PAGE.
• Publications:
  Neuraminidases 1 and 3 Trigger Atherosclerosis by Desialylating Low‐Density Lipoproteins and Increasing Their Uptake by Macrophages (2021)

Gene Information

• Gene Name: NEU1 sialidase 1 (lysosomal sialidase) [ Homo sapiens ]
• Official Symbol: NEU1
• Synonyms: NEU1; sialidase 1 (lysosomal sialidase); NEU; sialidase-1; G9 sialidase; exo-alpha-sialidase; lysosomal sialidase; acetylneuraminyl hydrolase; N-acetyl-alpha-neuraminidase 1; NANH; SIAL1; FLJ93471
• Gene ID: 4758
• mRNA Refseq: NM_000434
• Protein Refseq: NP_000425
• MIM: 608272
• UniProt ID: Q99519
• Chromosome Location: 6p21
• Pathway: Glycosphingolipid metabolism, organism-specific biosystem; Lysosome, organism-specific biosystem; Lysosome, conserved biosystem; Metabolism, organism-specific biosystem; Metabolism of lipids and lipoproteins, organism-specific biosystem; Other glycan degradation, organism-specific biosystem; Other glycan degradation, conserved biosystem
• Function: exo-alpha-(2->3)-sialidase activity; exo-alpha-(2->6)-sialidase activity; exo-alpha-(2->8)-sialidase activity; exo-alpha-sialidase activity; hydrolase activity, acting on glycosyl bonds

Citation

Neuraminidases 1 and 3 Trigger Atherosclerosis by Desialylating Low‐Density Lipoproteins and Increasing Their Uptake by Macrophages

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease    PubMed ID: 33554615    Data: 2021/2/6

Authors: Ekaterina P. Demina, Victoria Smutova, Alexey V. Pshezhetsky

Article Snippet:Because production of active recombinant NEU1 requires mammalian cells, the human enzyme was expressed as a His‐tagged protein in HEK293 cells, transduced with a CathA‐IRES‐NEU1 lentivirus, and partially purified by affinity chromatography using HisPur Ni‐NTA (Thermo Fisher Scientific; 88222).Because production of active recombinant NEU1 requires mammalian cells, the human enzyme was expressed as a His‐tagged protein in HEK293 cells, transduced with a CathA‐IRES‐NEU1 lentivirus, and partially purified by affinity chromatography using HisPur Ni‐NTA (Thermo Fisher Scientific; 88222).. For lectin blotting experiments, commercially available purified recombinant human NEU1 (NEU1‐156H; Creative BioMart) was used.. Neuraminidase activity and inhibition assays were performed using 2′‐(4‐methylumbelliferyl)‐α‐ d ‐N‐acetylneuraminic acid and GM3 ganglioside as substrates, as described.Neuraminidase activity and inhibition assays were performed using 2′‐(4‐methylumbelliferyl)‐α‐ d ‐N‐acetylneuraminic acid and GM3 ganglioside as substrates, as described.

A , Liquid chromatography–mass spectrometry (LC‐MS) profiles of ApoB N‐glycans from neuraminidase‐treated and untreated human LDL. Chromatograms of fluorescently labeled N‐glycans cleaved from ApoB of human LDL from a healthy donor (black) and of LDL treated with recombinant human neuraminidase 1 (NEU1), neuraminidase 2 (NEU2), neuraminidase 3 (NEU3), and neuraminidase 4 (NEU4) enzymes (blue, red, green, and purple, respectively). Samples were resolved on an anion exchange column, where glycans with higher sialic acid content are eluted at later retention times. For all samples, the intensities were normalized to those of the corresponding peaks of asialo glycans (10–11 minutes retention time). Figure shows representative profiles of duplicate experiments. Quantitation of peak areas and glycan assignment is shown in Tables . Bar graph shows relative changes in the asialo‐side, monosialo‐side (mono), disialo‐side (di), and trisialo‐side (tri) N‐glycan profiles for the neuraminidase‐treated compared with untreated LDL on the basis of the analysis of the corresponding MS/MS fragmentation patterns. B , Neu5Ac‐containing glycopeptides identified on ApoB. Purified LDL was treated with recombinant NEU3 enzyme or a buffer control, and the ApoB glycoprotein was resolved by SDS‐PAGE, digested with proteinase K, and analyzed by LC‐MS/MS to identify glycopeptides. A map of the primary protein sequence is shown (bottom) with the α (blue) and β (red) domains highlighted. Arrows indicate predicted sites of N‐linked glycosylation, with the numbers of the glycosylated asparagine residues shown below the protein map. Diamonds indicate detected glycopeptides containing at least one sialic acid. Data from Harazono et al ⁷⁶ are shown for comparison. Identified glycopeptides are shown in Tables . C , ApoB in neuraminidase‐treated LDL shows reduced staining with Sambucus nigra lectin (SNL) and Maackia amurensis lectin (MAL)‐II lectins and is recognized by peanut ( Arachis hypogaea ) agglutinin (PNA) lectin, consistent with its desialylation. Purified human LDLs (10 μg) were incubated with human recombinant NEU1 (1 and 2 mU), NEU2 (1 mU), NEU3 (1 mU), or NEU4 (1 mU), and glycosylation of ApoB was analyzed by blotting with SNL, MAL‐II, or PNA lectin. Ponceau S–stained membranes were used as controls for protein loading. Cont indicates control; LDLR, LDL receptor; MW, molecular weight; and nLDL, native LDL. * P <0.05 compared with control in ANOVA test (n=3).

A through D , Accumulation of native LDL and desialylated LDL (desLDL) in the aortic root lesions of 16‐week‐old Apoe ?/? mice was studied 6 hours after systemic injection of 200 μg of labeled LDL or desLDL. Aortic root sections were stained with monoclonal anti–monocyte+macrophage antibody (MOMA‐2) ( A ), polyclonal rabbit antibody against ASGR1 ( B ), or monoclonal rabbit antibody against neuraminidase 1 (NEU1) ( C ). D , Alexa 594 fluorescence in root sections was analyzed by fluorescent confocal microscopy. Panels show typical images of aortic root from untreated mice and those treated with Alexa 594–labeled LDL or desLDL. Bar graphs present average fluorescence intensities of aorta wall quantified with ImageJ software. Plots show mean values±SD obtained with 6 untreated mice and 6 LDL‐treated and 8 desLDL‐treated male and female mice. * P <0.05 and ** P <0.01 compared with untreated and LDL‐treated mice in Kruskal‐Wallis test, followed by the Dunn multiple comparisons test. E , Desialylation does not affect LDL uptake by liver hepatocytes. Accumulation of native LDL and desLDL in the livers of mice was studied 6 hours after systemic injection of 200 μg of labeled LDL. Sectioned livers were analyzed by fluorescent confocal microscopy. Relative fluorescence intensities were measured by ImageJ software. Panels show typical images of mice treated with Alexa 488–labeled LDL or desLDL. Graphs present fluorescence intensities of hepatocytes (individual and mean values±SD, measured in 50 cells from 4 mice per group). **** P <0.0001 compared with untreated mice in Kruskal‐Wallis test, followed by the Dunn multiple comparisons test. ASGR indicates asialoglycoprotein receptor; and Dapi, 4′,6‐diamidino‐2‐phenylindole.

A , Size of fatty streaks is reduced in the aortic root of Apoe ?/? mice deficient in NEU1 and NEU3. Female Apoe ?/? mice (n=17) and Apoe ?/? mice deficient in NEU1 (n=9), NEU3 (n=17), or neuraminidase 4 (NEU4) (n=9) were euthanized at the age of 16 weeks. Atherosclerosis was analyzed by staining fatty streaks in the aortic root sections with Red Oil O. Microphotographs show representative images of aortic root sections. Bar=500 μm. The graph shows atherosclerotic lesion size in the aortic roots (μm 2 ) measured by ImageJ software. * P <0.05 and ** P <0.01 compared with Apoe ?/? mice in Kruskal‐Wallis test, followed by the Dunn multiple comparisons test. B , Macrophage infiltration is reduced in atherosclerotic aortic root lesions of Apoe ?/? mice deficient in NEU1. Macrophage infiltration was studied in 16‐week‐old Apoe ?/? (n=8), Apoe ?/? CathA S190A‐Neo (n=9), Apoe ?/? Neu3 ?/? (n=10), and Apoe ?/? Neu4 ?/? (n=10) mice. Panels show representative photomicrographs of aortic root sections stained with anti–monocyte+macrophage antibody (MOMA‐2) antibodies. Bar=50 μm. The graph shows areas of MOMA‐2–positive area (μm 2 ) measured by ImageJ software. At least 4 sections per mouse were examined. Data represent mean±SD. * P <0.05 in Kruskal‐Wallis test, followed by the Dunn multiple comparisons test. C , Lipid plasma composition of 16‐week‐old female Apoe ?/? , Apoe ?/? Neu4 ?/? , Apoe ?/? Neu3 ?/? , and Apoe ?/? CathA S190A‐Neo mice. Total cholesterol, triglyceride, high‐density lipoprotein (HDL) cholesterol, and low‐density lipoprotein (LDL) cholesterol levels were measured in plasma samples. Data represent mean±SD. * P <0.05, ** P <0.01, and *** P <0.001 in 1‐way ANOVA test, followed by the Tukey multiple comparisons test. D , Ganglioside composition of plasma from 16‐week‐old female Apoe ?/? , Apoe ?/? Neu4 ?/? , Apoe ?/? Neu3 ?/? , and Apoe ?/? CathA S190A‐Neo mice. Values show mean±SD (n=3). The major glycan observed in all samples is GM2 containing a Neu5Gc residue (3–3.5 μg/mL). ⁵⁰ Other minor gangliosides are GM1, GA2, GM3 containing Neu5Gc, GM3, and LacCer. * P <0.05, ** P <0.01, and **** P <0.0001 compared with Apoe ?/? mice in Kruskal‐Wallis test, followed by the Dunn multiple comparisons test. E , Increased sialylation of LDL apolipoprotein B 100 (ApoB) in the blood of CathA S190A‐Neo mice. Blood was collected by cardiac puncture into EDTA‐coated tubes from Apoe ?/? , Apoe ?/? Neu4 ?/? , Apoe ?/? Neu3 ?/? , and Apoe ?/? CathA S190A‐Neo female 16‐week‐old mice (7–10 animals/group). For each group, LDL (d=1.019 to 1.063 g/mL) was isolated from 4 mL of pooled plasma by sequential density gradient ultracentrifugation. Sialylation of the ApoB was analyzed by lectin blotting using biotinylated Sambucus nigra lectin (SNL). The panel shows images of representative blots and corresponding membranes stained for protein by Ponceau S or Pierce Reversible Protein Stain. The graph shows results of quantification (SNL staining normalized for protein band intensity; mean values±SD) performed for 3 individual blots by ImageJ software. ** P <0.01 compared with wild type (WT) in Kruskal‐Wallis test, followed by the Dunn multiple comparisons test. F , LDL levels are increased in the plasma of Neu1 knockout (KO) mice. LDL cholesterol levels were measured in plasma samples of 8‐week‐old C57Bl6J (WT) and NEU1‐deficient CathA S190A‐Neo mice, as well as Neu1 KO Neu1 ENSMUSE141558 and Neu1 ΔEx3 mice. Data represent mean±SD. **** P <0.001 compared with WT mice in 1‐way ANOVA test, followed by the Tukey multiple comparisons test.