Maria O. Longas

Sulfate composition of glycosaminoglycans determined by infrared spectroscopy

Anhydrous sodium sulfate (Na2SO4) was analyzed at varying concentrations by infrared (ir) spectroscopy. A standard curve was obtained from a linear plot of sulfate (SO2-(4] concentration vs the weight of the ir band area of S = O stretching. Standard chondroitin 4-sulfate, chondroitin 6-sulfate, heparan sulfate, heparin, keratan sulfates, and various dermatan sulfates isolated from human and rat skins were also studied by ir spectroscopy. The spectrum of every glycosaminoglycan (GAG) displayed an ir band around 1230 cm-1 which originated from S = O stretching of sulfate esters. Therefore, the weight of the latter band was employed to quantify sulfate, by using the standard curve indicated above. Sulfate was also estimated quantitatively by the gelatin/BaCl2 method of K.S. Dodgson and R.G. Price (Biochem. J. 1962, 84, 106-110). The sulfate composition determined by ir spectroscopy ranged from 8.5 to 22.1% (w/w), and agreed closely with the values obtained chemically. In the ir spectroscopy method, sulfate was determined using the polymer forms of the GAGs. After analysis, these heteropolysaccharides were recovered unaffected in a yield greater than 95%. The data show that the infrared spectroscopy technique, in addition to being sensitive and reliable, is much more economical than the chemical procedures currently employed to quantify GAG sulfate.

A tightly bound protein-porphyrin complex isolated from the plasma of a patient with variegate porphyria

Free acid porphyrins were isolated from plasma of a patient with variegate porphyria. Part of the total porphyrin content--which included protoporphyrin IX, harderoporphyrin and uroporphyrin in a molar ratio of 1.2:1:0.5 and traces of pentacarboxylic porphyrin--was extractable with ethyl acetate/acetic acid as described previously [1]. Unextractable porphyrins remained in the precipitate formed after mixing the lower liquid layer and precipitate from the extraction procedure (Fig. 1, [1]) with excess ethyl acetate/acetic acid. A portion of this precipitate was hydrolyzed in 8 mol/l HCl; its porphyrins were extracted with N-butanol and analyzed by high pressure liquid chromatography; another portion was chromatographed on Sephadex G-150 with 1 mol/l MgCl2, and the major porphyrin-protein pool was hydrolyzed in 8 mol/l HCl, reacted separately with AgNO3 and Ag2SO4, and subjected to cellulose acetate and polyacrylamide-gel electrophoresis. The results support the hypothesis that a dicarboxylic porphyrin, a major portion of which was unextractable by standard procedures [1] and which appeared to be covalently bound to a protein of approximately 68 000 mol. wt. that moved with human serum albumin during cellulose acetate electrophoresis, is the preponderant porphyrin in this plasma.

Dietary vitamin E reverses th effects of ultraviolet light irradiation on rat skin glycosaminoglycans

Chondroitin 4-sulfate (Ch 4-S), three dermatan sulfates (DS18, DS45a, DS45b) and hyaluronic acid (HA) were the major glycosaminoglycans (GAG) isolated from the skin of 4 groups of albino rats. The yields from Group 1 (control) were: Ch 4-S, 0.015%; HA, 0.028% and DS (total), 0.098% (w/w). Traces of heparin were detected only in rats irradiated with ultraviolet (UV) light (Group II), in the GAG pool isolated with 45% ethanol. Yields increased by at least 28% (w/w) in Group II, but decreased, except HAs, also by at least 28%, below the level of the control, in irradiated rats that also ingested vitamin E (Group III). The sulfate composition of these GAG determined by infrared spectroscopy was as follows: approx. 17% (w/w) for DS18, 21-30% for DS45a, 21-35% for DS45b and 26-44% for Ch 4-S. A 60-70% (mol/mol) N-acetylation of hexosamine in DS45 was estimated by Fourier transform 1H-NMR spectroscopy; the IdUA composition of this DS was 30-46% (mol/mol), and the uronic acid/hexosamine ratio ranged from 2.50:1 to 1.6:1. The data show UV light irradiation of rat skin to result in an abnormally elevated production of the major GAG and oversulfation of Ch 4-S and DS. These effects are reversed, except for the sulfation of DS45b, when the irradiated animals also ingest vitamin E.

Heparin antiproliferative activity on bovine pulmonary artery smooth muscle cells requires both N-acetylation and N-sulfonation

The antiproliferative activity of Heparin (HP) on bovine pulmonary artery smooth muscle cells (BPASMC) in vitro requires both N-acetylation and N-sulfonation. This was demonstrated by quantifying the relative N-acetylation of three commercial heparins of known antiproliferative activities, using their Fourier-transform infrared (FTIR) band areas at 1381-1378 and 1320-1317 cm(-1), which combined resulted in 1.0, 1.0 and 1.3 cm2 for Choay, Elkins-Sinn and Upjohn HP, respectively. These results show that Upjohn HP, which is at least 44% more antiproliferative than the other two, is 30% more N-acetylated. Upjohn HP was also N-desulfonated chemically, and its antiproliferative activity was determined. Its total sulfonate (--SO 3 -) content (O- and N-sulfonate) was quantified using the FTIR band area at 1260-1200 cm(-1) for the S=O stretching; a drop in sulfonate content from 21.87% (w/w) before N-desulfonation to 16.51% (w/w) after N-desulfonation, resulted in a 67% decrease in its inhibitory potency. In addition to the requirement that approximately 24% of the sulfonate content be bonded to N, the data show a direct correlation between the extent of Upjohn HP N-acetylation and its antiproliferative activity on BPASMC.

Partial characterization of dermatan sulfate by proton nuclear magnetic resonance spectroscopy

The degree of galactosamine N-acetylation, iduronic acid composition, and total uronic acid/hexosamine ratios of the three dermatan sulfates of human skin, DS18, DS28, and DS35 (M. O. Longas et al. (1987) Carbohydr. Res. 159, 127-136), were determined by Fourier transform, proton nuclear magnetic resonance (FT 1H NMR) spectroscopy. Analysis of DS of varying ages was conducted at 400 MHz and 60 degrees C. Chemical shifts for H-1, H-2, H-4, and H-5 of L-IdUA were independent of those for the respective protons of D-GalNAc and D-GlcUA. The resonance intensities of H-1 and acetamido methyl protons of D-GalNac did not display the expected 1:3 ratios. Therefore, their integration values were employed to estimate the percentage N-acetylation (N-CH3/3 H-1) which was corroborated chemically. The L-IdUA content, relative to total uronic acid, was calculated from signal intensities of H-1 of L-IdUA and D-GlcUA and ascertained by quantitative chemical methods. Total uronic acid/hexosamine ratios were determined from both 1H NMR spectroscopy and chemical analyses. The data show the following N-acetylation (N-CH3/3 H-1) of galactosamine in DS:DS18, 61-72% between 17 and 60 years, unaffected by senescence; DS28, 78-86% with no age-related trend; DS35, 101% at 19 years. Furthermore, in all ages investigated, the percentage (wt/wt) L-IdUA relative to total uronic acid was 42-44% for DS18 and 37-40% for DS28. At age 19 years, DS35 had a 29% (wt/wt) L-IdUA. The total uronic acid/hexosamine ratios for DS18 and DS28 varied from 1.40:1.0 to 1.70:1.0 irrespective of age.

N-Acetylglucosaminyl 1-Phosphate Transferase: An Excellent Target for Developing New Generation Breast Cancer Therapeutic

Studies from our laboratory have explained that breast tumor progression can be attenuated by targeting the N-linked glycoproteins of the tumor microvasculature and that of tumor cells alike with a protein N-glycosylation inhibitor, tunicamycin. Absence of N-glycosylation leads to an accumulation of un- or mis-folded proteins in the ER and the cell develops “ER stress”. The result is cell cycle arrest, and induction of apoptosis mediated by unfolded protein response (upr) signaling. Tunicamycin inhibited in vitro and in vivo (Matrigel™ implants in athymic nude mice) angiogenesis in a dose dependent manner. The action is irreversible and survived under tumor microenvironment, i.e., in the presence of FGF-2 or VEGF or higher serum concentration. Importantly, tunicamycin prevented the progression of double negative (ER−/PR−/Her2+) and triple negative (ER−/PR−/Her2−) breast tumors by ~55 to 65 % in 3 weeks in athymic nude mice [Balb/c(nu/nu)]. Analyses of paraffin sections exhibited “ER stress” in both microvasculature and in tumor tissue.

Balancing life with glycoconjugates: monitoring unfolded protein response-mediated anti-angiogenic action of tunicamycin by Raman Spectroscopy

Asparagine-linked protein glycosylation is a hallmark for glycoprotein structure and function. Its impairment by tunicamycin [a competitive inhibitor of N-acetylglucos-aminyl 1-phosphate transferase (GPT)] has been known to inhibit neo-vascularization (i.e., angiogenesis) in humanized breast tumor due to an induction of endoplasmic reticulum (ER) stress-mediated unfolded protein response (UPR). The studies presented here demonstrate that (i) tunicamycin inhibits capillary endothelial cell proliferation in a dose-dependent manner; (ii) treated cells are incapable of forming colonies upon its withdrawal; and (iii) tunicamycin treatment causes nuclear fragmentation. Tunicamycin-induced ER stress-mediated UPR event in these cells was studied with the aid of Raman spectroscopy, in particular, the interpretation of bands at 1672, 1684, and 1694 cm–1, which are characteristics of proteins and originate from C=O stretching vibrations of mono-substituted amides. In tunicamycin-treated cells, these bands decreased in area as follows: at 1672 cm–1 by 41.85 % at 3 h and 55.39 % at 12 h; at 1684 cm–1 by 20.63 % at 3 h and 40.08 % at 12 h; and also at 1994 cm–1 by 33.33 % at 3 h and 32.92 % at 12 h, respectively. Thus, in the presence of tunicamycin, newly synthesized protein chains fail to arrange properly into their final secondary and/or tertiary structures, and the random coils they form had undergone further degradation.

Chapter 17 – Hyaluronan in Aging

Hyaluronan (HA) is an essential heteropolysaccharide of mammalian connective tissue. In this chapter, the secondary and tertiary structures of HA in solution are analyzed, in an attempt to explain their relevance to normal biologic functions and aging. Hydrophilic sequences of D-GlcN and D-GlcA are used to explain the stiff and flexible domains detected in HA solutions. The hydrophilic segments should be more densely hydrogen bonded with water than the regions of alternating D-GlcA and D-GlaNAc, as water would form cages around them. In aging, the hypothetic D-GlcN sequence is used in an attempt to explain the N-deacetylation of human skin HA at 75 years of age. Because the skin composition of HA decreases by 77% (w/w), also at 75 years, and the basic sugarsequence makes HA more hydrophilic than the alternating sequence of D-GlcA and D-GlcNAc, it is concluded that nature compensates for the loss of HA concentration in aging by having an enzyme that removes the N-acetyl groups, making HA more hydrophilic. Highly hydrophilic HA is needed in aged human skin, if no other molecules are made to retain the water, since about 50% (w/w) of this reagent is bonded to this GAG. The reasons for the loss of HA from the skin were attributed to depolymerization induced by both endogenous and exogenous free radicals. This hypothesis is supported by findings that indicate that free radical scavengers inhibit UV-light-mediated cleavage of HA in vitro.

Abstract 5074: Anti-angiogeneic glycotherapy tunicamycin induces ER stress mediated unfolded protein response in beast cancer cells.

Tumor growth is angiogenesis dependent. Most of the current therapeutics targets either the tumor cells or the microvasculature but not necessarily the both. As a consequence, the patients enjoy disease free survival only for a limited period of time, irrespective of a good initial prognosis and pathological response rates. Aberrant glycosylation has been observed in experimental and human tumors and we have hypothesized that a good asparagine-linked (N-linked) protein glycosylation inhibitor could target both endothelial cells and tumor cells and eliminate the tumor progression. Our objective is therefore to test the efficacy of such an inhibitor, tunicamycin. The results support that tunicamycin inhibits angiogenesis in vitro and in vivo, and the breast tumor microvasculature. There is cell cycle arrest followed by apoptosis due to ER (endoplasmic reticulum)stress-mediated unfolded protein response (upr). To evaluate the intracellular protein status under upr, the proteome was analyzed by Raman spectroscopy focusing on bands at 1672, 1684 and 1694 cm−1, which are characteristics of proteins and originate from C=O stretching vibrations of mono-substituted amides. In tunicamycin-treated cells these bands decreased in area. Thus, in the presence of tunicamycin, newly synthesized protein chains fail to arrange properly into their final secondary and/or tertiary structures, and the random coils they form had undergone further degradation. Tunicamycin treatment (i) prevents the colony formation, and (ii) fails to reverse the inhibition of angiogenesis by VEGF165. VEGF-specific protein tyrosine phosphorylation in capillary endothelial cells is also inhibited. When tested, tunicamycin indiscriminately inhibited the progression of a double negative (MDA-MB-435) and a triple negative (MDA-MB-231) breast cancer by ∼55-65% in three weeks in athymic nude mice, Balb/c(nu/nu). High GRP-78/Bip expression evidenced ER stress in tumor tissue. To evaluate the specificity and efficacy of tunicamycin on tumor cells, five different breast cancer cell lines with distinct genotypic and phenotypic characteristics were analyzed. Cellular proliferation of MDA-MB-231, MDA-MB-468, BT-20, BT-474 and ZR-731 cells is inhibited almost to the same extent and the cells also failed to colonize. High protein and mRNA levels for GRP-78/Bip in MDA-MB-231 breast cancer cells indicate ER stress. Down regulation of cyclin D1 expression indicates cell cycle impairment irrespective of high F2F1, Bcl-2 and phospho-p53 expression with no change in CDK4. Total Rb is increased in tunicamycin treated cells but pRb is down-regulated. Cell death due to apoptosis was evidenced and concluded that upr signaling causes a translational attenuation in MDA-MB-231 breast cancer cells. Supported in part by grants from Susan G. Komen for the Cure BCTR0600582 (DKB) and NIH/NIMHD 8G12MD007583 (KB). Citation Format: Dipak K. Banerjee, Aditi Banerjee, Lydia Espinoza, Neyshari Sanchez, Krishna Baksi, Maria O. Longas. Anti-angiogeneic glycotherapy tunicamycin induces ER stress mediated unfolded protein response in beast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5074. doi:10.1158/1538-7445.AM2013-5074

Human skin dermatan sulfate with sulfated and unsulfated non-reducing ends

The non-reducing ends of the preponderant dermatan sulfates of adult human skin (DS18 and DS28) can have D-GalNAc, D-GlcA and L-1doA. D-GlcA of DS18 and D-GalNAc of both DS18 and DS28 are sulfated.