Journal: Scientific Reports

Article Title: Structure and functional impact of glycosaminoglycan modification of HSulf-2 endosulfatase revealed by atomic force microscopy and mass spectrometry

doi: 10.1038/s41598-023-49147-5

Figure Lengend Snippet: Mass spectrometry and NMR identification of the CS/DS GAG chain attached to HSulf-2. ( a ) Negative reflector MALDI-TOF analysis of CS oligosaccharides from digestion of HSulf-2 by chondroitinase ABC. Mass spectra in the 380–800 m / z range corresponding to dp2 (top), the 800–1600 m / z range corresponding to dp4 (middle), and the 1400–2000 m / z range corresponding to dp6 and dp8 (bottom). M is the molecular ion corresponding to the fully sodiated sulfated oligosaccharide: ΔDP8, 4S, 4Ac, 8Na, M = 2012.13 Da; ΔDP6, 3S, 3Ac, 6Na, M = 1509.10 Da; ΔDP4, 2S, 2Ac, M = 1006.06 Da. (oligosaccharide samples were mixed in a 1:1 volume ratio with the ionic liquid matrix 9 mg/mL HABA/TMG 2 doped with 100 µM NaCl; * = ions from matrix). ( b ) HILIC-MS of disaccharides released from HSulf-2 by the chondroitinase ABC: Separation of (top) CS disaccharide standards A, C, D, E, and non sulfated CS-0 (EIC of CS-0 in light grey at m / z 378; EIC of monosulfated CS-C and CS-A at m / z 458.1 in black; EIC of disulfated CS-D, CS-E and CS-2,4 at m / z 538.1 in dark grey), and (bottom) disaccharides released from HSulf-2 by the chondroitinases ABC. ( c ) NMR analysis of the CS/DS oligosaccharides released from HSulf-2 by hyaluronidase depolymerization. Proton chemical shifts (ppm)-based identification of sulfated GalNAc, GlcA and IdoA residues from CS/DS oligosaccharides released from HSulf-2 by hyaluronidase (Left table). Methyl group region of the 1D 1H spectrum evidencing the presence of both 4- O - and 6- O -sulfated GalNAc residues in CS/DS oligosaccharides. ( d ) CID fragmentation spectrum of the glycopeptide 542 HWPGAPEDQDDKDGGDFSGTGGLPDYSAANPIK 574 identified by nanoLC–MS/MS analysis as carrying the tetrasaccharide attaching the CS/DS chain to HSulf-2. The selected precursor is m / z 1122.6935 (4 +). M: mass of the whole glycopeptide; Mpep: mass of naked backbone peptide; dehydrated glucuronic acid (dGlcA): ; N -acetylgalactosamine (GalNAc): ; dehydrated N-acetylgalactosamine (dGlcNAc): ; monosulfated-N-acetylgalactosamine (GalNAcS): ; Galactose: ; dehydrated galactose: ; Glucuronic acid (GlcA): ; Xylose:

Article Snippet: The recombinant human hyaluronidase 4/HYAL4 (6904-GH) was from R&D Systems (Bio-Techne, France).

Techniques: Mass Spectrometry, Hydrophilic Interaction Liquid Chromatography, Tandem Mass Spectroscopy

Journal: International Journal of Molecular Sciences

Article Title: Lysosome Alterations in the Human Epithelial Cell Line HaCaT and Skin Specimens: Relevance to Psoriasis

doi: 10.3390/ijms20092255

Figure Lengend Snippet: Real-time qRT-PCR verification of selected genes related to the microphthalmia family (MiT family) (Panel A: MITF , TFE3 , TFEB , and TFEC ) ; lysosomal marker genes and those encoding factors that control the lysosomal biogenesis process (Panel B: LAMP1 , MCOLN1 , MTORC1 , PPP3CA , and PPP3CB ); genes encoding lysosomal enzymes (Panel C: ASAH , GLA , GM2A , HPSE , HYAL4 , PSAP , SMPD1 , and SPHK1 ); and selected markers for psoriasis ( IVL , S100A7 , and S100A9 ) in the human epithelial cell model of HaCaT cells with “psoriasis-like” inflammation. The relative mRNA expression levels were determined against TBP (TATA-Box Binding Protein) in “cytokine mix”-activated HaCaT cells cultured in medium containing Ca 2+ ≤ 0.1 mM vs. the non-activated HaCaT cell control cultured in medium containing either ≤0.1 or 2 mM Ca 2+ , and the relative quantitation was calculated using 2 −ΔΔ C t (FC—fold change). Bold font signifies a change in the expression level (≥1.3 and ≤0.7, respectively). Data represent the mean values ± standard deviation (SD) from n = 3; the non-activated HaCaT cell control cultured in medium containing a Ca 2+ concentration ≤0.1; “cytokine mix”-activated HaCaT cells cultured in medium containing 2 mM Ca 2+ ; and the non-activated HaCaT cell control cultured in medium containing 2 mM Ca 2+ .

Article Snippet: The TaqMan probes used were as follows: ASAH (Hs00602774_m1), GAPDH (Hs02786624_g1), GLA (Hs00609238_m1), GM2A (Hs00166197_m1), HPSE (Hs00935036_m1), HYAL4 (Hs00202177_m1), IVL (Hs00846307_s1), LAMP1 (Hs00931461_m1), MCOLN1 (Hs01100653_m1), MITF (Hs01117294_m1), MTORC1 (Hs00234508_m1), PPP3CA (Hs00174223_m1), PPP3CB (Hs00236113_m1), PSAP (Hs01551096_m1), S100A7 (Hs01923188_u1), S100A9 (Hs00610058_m1), SMPD1 (Hs03679346_g1), SPHK1 (Hs00184211_m1), TBP (Hs00427620_m1), TFE3 (Hs00232406_m1), TFEB (Hs00292981_m1), and TFEC (Hs00992838_m1).

Techniques: Marker, Control, Expressing, Binding Assay, Cell Culture, Quantitation Assay, Standard Deviation, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Lysosome Alterations in the Human Epithelial Cell Line HaCaT and Skin Specimens: Relevance to Psoriasis

doi: 10.3390/ijms20092255

Figure Lengend Snippet: Real-time qRT-PCR verification of microphthalmia family (MiT family) genes ( A : MITF , TFE3 , TFEB , and TFEC ); lysosomal marker genes and those encoding factors that control the lysosomal biogenesis process ( B : LAMP1 , MCOLN1 , MTORC1 , PPP3CA , and PPP3CB ); and genes encoding lysosomal enzymes ( C : ASAH , GLA , GM2A , HPSE , HYAL4 , PSAP , SMPD1 , and SPHK1 ) in skin specimens. The relative mRNA expression levels were determined against that of GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and the relative quantitation was calculated using 2 −ΔΔ C t and compared between each group (PP—psoriatic plaque vs. NN—non-psoriatic normal, PN—psoriatic normal vs. NN, and PP vs. PN). Scatter plots showing the relative expression levels of genes, each with number of patients n = 11 (psoriasis patients 8 to 18) for PP/PN and n = 11 (healthy individuals 5 to 15 as a control) for NN.

Article Snippet: The TaqMan probes used were as follows: ASAH (Hs00602774_m1), GAPDH (Hs02786624_g1), GLA (Hs00609238_m1), GM2A (Hs00166197_m1), HPSE (Hs00935036_m1), HYAL4 (Hs00202177_m1), IVL (Hs00846307_s1), LAMP1 (Hs00931461_m1), MCOLN1 (Hs01100653_m1), MITF (Hs01117294_m1), MTORC1 (Hs00234508_m1), PPP3CA (Hs00174223_m1), PPP3CB (Hs00236113_m1), PSAP (Hs01551096_m1), S100A7 (Hs01923188_u1), S100A9 (Hs00610058_m1), SMPD1 (Hs03679346_g1), SPHK1 (Hs00184211_m1), TBP (Hs00427620_m1), TFE3 (Hs00232406_m1), TFEB (Hs00292981_m1), and TFEC (Hs00992838_m1).

Techniques: Quantitative RT-PCR, Marker, Control, Expressing, Quantitation Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: A Novel Splice Variant of HYAL-4 Drives Malignant Transformation and Predicts Outcome in Bladder Cancer Patients.

doi: 10.1158/1078-0432.CCR-19-2912

Figure Lengend Snippet: A - D: Transcript levels of hyaluronidase gene family members in bladder tissues. Normal bladder: NBL (n=31); low-grade BC: LG (n=7); high-grade non-muscle invasive BC: HG NMIBC (n=6); HG MIBC (n=39). (A): HYAL-2, HYAL-3, PH20; (B): HYAL-4 (C): HYAL-4 Wt; (D): V1. E: V1 levels of HG MIBC specimens further stratified by clinical outcome (metastasis and CSS). Data: Mean ± SEM. P-values two-tailed; Mann-Whitney U test. F and G: Kaplan-Meier plot showing risk-stratification of the cohort by V1 mRNA levels for metastasis and CSS.

Article Snippet: HYAL-4 shRNA transfectants: HYAL-4 shRNA constructs were purchased from Origene.

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: A Novel Splice Variant of HYAL-4 Drives Malignant Transformation and Predicts Outcome in Bladder Cancer Patients.

doi: 10.1158/1078-0432.CCR-19-2912

Figure Lengend Snippet: A: Immunoblot analysis of bladder tissue extracts using anti-HYAL-4 antibody. B: Chase levels (U/mg protein) in bladder tissue extracts measured by Chase assay. C: HYAL-4 staining in representative NBL and BC tissues. Magnification: 400X. See Figure S2 for enlarged photos and quantification. D: Immunoblot anlaysis of cell lysates and CM of EV, Wt, and V1, Ctr, and HYAL-4 shRNA transfectants for indicated proteins; loading control: Actin. HYAL-4 peptide block: the peptide was added during incubation with the anti-HYAL-4 antibody. Note for A and D: In each blot, all sample lanes were run on the same gel with the same exposure time; a gap denotes those samples that were not contiguous within the gel. E: Chase activity (U) was measured in the CM of bladder cell transfectants (left panel) and in the CM of control (Ctr) and HYAL-4 shRNA transfectants (right panel). The activity was normalized to cell number. Data: Mean ± SEM; quadruplicate. F: Normalized Chase activity in the CM of HT1376 transfectants and in a high-grade bladder tumor tissue extract (HG-TBL) at indicated pH. Data: Mean ± SEM; quadruplicate. G: Chase activity in the CM of HT1376 V1 transfectants and a HG-TBL extract measured against different chondriotin sulfate substrates. Data: Mean ± SEM; quadruplicate.

Article Snippet: HYAL-4 shRNA transfectants: HYAL-4 shRNA constructs were purchased from Origene.

Techniques: Western Blot, Staining, shRNA, Blocking Assay, Incubation, Activity Assay