Hyperprolactinemia modifies extracellular matrix components associated with collagen fibrillogenesis in harderian glands of non- and pregnant female mice.

The harderian gland (HG) is a gland located at the base of the nictating membrane and fills the inferomedial aspect of the orbit in rodents. It is under the influence of the hypothalamic-pituitary-gonadal axis and, because of its hormone receptors, it is a target tissue for prolactin (PRL) and sex steroid hormones (estrogen and progesterone). In humans and murine, the anterior surface of the eyes is protected by a tear film synthesized by glands associated with the eye. In order to understand the endocrine changes caused by hyperprolactinemia in the glands responsible for the formation of the tear film, we used an animal model with metoclopramide-induced hyperprolactinemia (HPRL). Given the evidences that HPRL can lead to a process of cell death and tissue fibrosis, the protein expression of small leucine-rich proteoglycans (SLRPs) was analyzed through immunohistochemistry in the HG of the non- and the pregnant female mice with hyperprolactinemia. The SRLPs are related to collagen fibrillogenesis and they participate in pro-apoptotic signals. Our data revealed that high prolactin levels and changes in steroid hormones (estrogen and progesterone) can lead to an alteration in the amount of collagen, and in the structure of type I and III collagen fibers through changes in the amounts of lumican and decorin, which are responsible for collagen fibrillogenesis. This fact can lead to the impaired functioning of the HG by excessive apoptosis in the HG of the non- and the pregnant female mice with HPRL and especially in the HG of pregnancy-associated hyperprolactinemia.

Hyaluronic acid and proliferation/cellular death amount in the female rats mammary gland after estroprogestative therapy.

AIMS: To evaluate the concentration of hyaluronan acid and proliferation/cellular death in mammary gland of ovariectomized female rat after estroprogestative therapy. MATERIALS AND METHODS: Forty ovariectomized female rats were divided into four groups with 10 animals/each: OG (vehicle); EG: (Estradiol, 7 days of treatment), PG (Progesterone acetate, 23 days of treatment), and EPG: (Estradiol, 7 days of treatment, and next Progesterone acetate, 23 days of treatment). Twenty-four hours after the last treatment, all animals were euthanized, the mammary gland removed, then, a fragment was immersed in acetone to quantifying of the hyaluronan acid biochemical method (ELISA-Like fluorometric assay), and a fragment fixed for 24 h in 10% formaldehyde in phosphate-buffered saline (PBS) processed for immunohistochemistry method for detection of the cell marker proliferation (Ki67) and cellular marker death by DNA fragmentation the TUNEL method. RESULTS: The estradiol-treatment alone (EG) or associated with progesterone (EPG) affected the concentration of hyaluronan acid, increased cell proliferation, and decreased cell death compared to OG and PG (p < .05) in the mammary tissue. CONCLUSIONS: Our results suggest that the excessive reduction of HA in mammary tissue, as occurred with progesterone treatment, can lead to a breakdown of the extracellular matrix. These changes may be indicative of mammary pathology such as the development of tumor.

Neuroprotective effect of heparin Trisulfated disaccharide on ischemic stroke.

Cells undergoing hypoxia experience intense cytoplasmic calcium (Ca2+) overload. High concentrations of intracellular calcium ([Ca2+]i) can trigger cell death in the neural tissue, a hallmark of stroke. Neural Ca2+ homeostasis involves regulation by the Na+/Ca2+ exchanger (NCX). Previous data published by our group showed that a product of the enzymatic depolymerization of heparin by heparinase, the unsaturated trisulfated disaccharide (TD; ΔU, 2S-GlcNS, 6S), can accelerate Na+/Ca2+ exchange via NCX, in hepatocytes and aorta vascular smooth muscle cells. Thus, the objective of this work was to verify whether TD could act as a neuroprotective agent able to prevent neuronal cell death by reducing [Ca2+]i. Pretreatment of N2a cells with TD reduced [Ca2+]i rise induced by thapsigargin and increased cell viability under [Ca2+]I overload conditions and in hypoxia. Using a murine model of stroke, we observed that pretreatment with TD decreased cerebral infarct volume and cell death. However, when mice received KB-R7943, an NCX blocker, the neuroprotective effect of TD was abolished, strongly suggesting that this neuroprotection requires a functional NCX to happen. Thus, we propose TD-NCX as a new therapeutic axis for the prevention of neuronal death induced by [Ca2+]i overload.

Diagnostic Accuracy of Serum Hyaluronan for Detecting HCV Infection and Liver Fibrosis in Asymptomatic Blood Donors.

Background: The disease caused by hepatitis C virus (HCV) is asymptomatic, silent, and progressive liver disease. In HCV-infected patients the increase in serum HA is associated with the development of hepatic fibrosis and disease progression. Methods: HCV-RNA detection was performed in all serological samples of blood donors that tested positive using HCV Ultra ELISA. Determination of hyaluronan (HA) was performed in positive HCV samples using ELISA-like fluorometric method. The HA content was compared to HCV viral load, genotype of the virus, liver fibrosis as well as ALT and GGT liver biomarkers. Results: Persistently normal ALT (<40 U/L) and GGT (<50 U/L) serum levels were detected in 75% and 69% of the HCV-Infected blood donors, respectively. Based on ROC analysis, the HA value < 34.2 ng/mL is an optimal cut-off point to exclude HCV viremia (specificity = 91%, NPV = 99%). Applying HA value ≥34.2 ng/mL significant liver fibrosis (≥F2) can be estimated in 46% of the HCV-infected blood donors. HA serum level (≥34.2 ng/mL) associated with a high ALT level (>40 U/mL) can correctly identify HCV infection and probable liver fibrosis (sensitivity = 96% and specificity = 90%) in asymptomatic blood donors. Conclusions: A high level of HA (≥34.2 ng/mL) in association with ALT (≥40 U/L) in serum can provide a good clinical opportunity to detect HCV-infected asymptomatic persons that potentially require a liver biopsy confirmation and antiviral treatment to prevent the development of advanced liver fibrosis or cirrhosis.

Crude Heparin Preparations Unveil the Presence of Structurally Diverse Oversulfated Contaminants.

Nowadays, pharmaceutical heparin is purified from porcine and bovine intestinal mucosa. In the past decade there has been an ongoing concern about the safety of heparin, since in 2008, adverse effects associated with the presence of an oversulfated chondroitin sulfate (OSCS) were observed in preparations of pharmaceutical porcine heparin, which led to the death of patients, causing a global public health crisis. However, it has not been clarified whether OSCS has been added to the purified heparin preparation, or whether it has already been introduced during the production of the raw heparin. Using a combination of different analytical methods, we investigate both crude and final heparin products and we are able to demonstrate that the sulfated contaminants are intentionally introduced in the initial steps of heparin preparation. Furthermore, the results show that the oversulfated compounds are not structurally homogeneous. In addition, we show that these contaminants are able to bind to cells in using well known heparin binding sites. Together, the data highlights the importance of heparin quality control even at the initial stages of its production.

Heparan sulfate proteoglycan deficiency up-regulates the intracellular production of nitric oxide in Chinese hamster ovary cell lines.

We investigated the role of glycosaminoglycans (GAGs) in the regulation of endothelial nitric oxide synthase (eNOS) activity in wild-type CHO-K1 cells and in xylosyltransferase-deficient CHO-745 cells. GAGs inhibit the integrin/FAK/PI3K/AKT signaling pathway in CHO-K1 cells, decreasing the phosphorylation of eNOS at Ser1177. Furthermore, in CHO-K1 cells, eNOS and PKCα are localized at sphingolipid- and cholesterol-rich domains in the plasma membrane called caveolae. At caveolae, PKCα activation stimulates the phosphorylation of eNOS on Thr495, resulting in further inhibition of NO production in these cells. In our data, CHO-745 cells generate approximately 12-fold more NO than CHO-K1 cells. Increased NO production in CHO-745 cells promotes higher rates of protein S-nitrosylation and protein tyrosine nitration. Regarding reactive oxygen species (ROS) production, CHO-745 cells show lower basal levels of superoxide (O2- ) than CHO-K1 cells. In addition, CHO-745 cells express higher levels of GPx, Trx1, and catalase than CHO-K1 cells, suggesting that CHO-745 cells are in a constitutive nitrosative/oxidative stress condition. Accordingly, we showed that CHO-745 cells are more sensitive to oxidant-induced cell death than CHO-K1 cells. The high concentration of NO and reactive oxygen species generated by CHO-745 cells can induce simultaneous mitochondrial biogenesis and antioxidant gene expression. These observations led us to propose that GAGs are part of a regulatory mechanism that participates in eNOS activation and consequently regulates nitrosative/oxidative stress in CHO cells.

Concentration of sulfated glycosaminoglycans in the mammary tissue of female rats with the aging and about hormonal influence.

It was to evaluate the concentration of sulfate glycosaminoglycans (GAG) in mammary tissue of the young and adult female rats and ovariectomized females rats after hormonal stimulation. For this purpose, 60 female rats were divided into six groups with 10 animals/each: nonovariectomized groups: G1 (5 months), and G2 (15 months) and ovariectomized groups: OG (vehicle); EG: (estradiol, 7 days of treatment), PG (progesterone acetate, 23 days of treatment) and EPG: (estradiol (7 days of treatment) and next progesterone acetate (23 days of treatment). Twenty-four hours after the last treatment, all animals were euthanized, the mammary tissue removed, processed for biochemical evaluation and quantification of the GAG. The comparison between groups showed that the concentration dermatan sulfate (DS) G1 was lower compared to G2, OG, EG (p < .05) and G2 was lower compared to OG (p < .05), and OG was higher compared to EG, GP, EPG (p < .05); and heparan sulfate (HS) G1 was higher compared to G2 (p < .05), and G2 was higher compared to OG, EP, PG and EPG (p < .05). These changes in the extracellular matrix might explain, at least in part, hormonal influence about sulfated glycosaminoglycans in response to physiological state/age, and in response to hormonal treatment in the mammary tissues.

CdSe magic-sized quantum dots incorporated in biomembrane models at the air-water interface composed of components of tumorigenic and non-tumorigenic cells.

Cadmium selenide (CdSe) magic-sized quantum dots (MSQDs) are semiconductor nanocrystals with stable luminescence that are feasible for biomedical applications, especially for in vivo and in vitro imaging of tumor cells. In this work, we investigated the specific interaction of CdSe MSQDs with tumorigenic and non-tumorigenic cells using Langmuir monolayers and Langmuir-Blodgett (LB) films of lipids as membrane models for diagnosis of cancerous cells. Surface pressure-area isotherms and polarization modulation reflection-absorption spectroscopy (PM-IRRAS) showed an intrinsic interaction between the quantum dots, inserted in the aqueous subphase, and Langmuir monolayers constituted either of selected lipids or of tumorigenic and non-tumorigenic cell extracts. The films were transferred to solid supports to obtain microscopic images, providing information on their morphology. Similarity between films with different compositions representing cell membranes, with or without the quantum dots, was evaluated by atomic force microscopy (AFM) and confocal microscopy. This study demonstrates that the affinity of quantum dots for models representing cancer cells permits the use of these systems as devices for cancer diagnosis.

Acquisition of anoikis resistance promotes alterations in the Ras/ERK and PI3K/Akt signaling pathways and matrix remodeling in endothelial cells.

Anoikis is a programmed cell death induced upon cell detachment from extracellular matrix. Anoikis resistance is a critical mechanism in tumor metastasis. Cancer cells deregulate and adapt their metabolism to survive in the absence of adhesion, spreading metastases to distant organs. These adaptations include abnormal regulation of growth factor receptors activating prosurvival signaling pathways, such as the Ras/ERK and PI3K/Akt pathways, and extracellular matrix remodeling, leading to metastasis by an increase of invasiveness and inhibiting anoikis. This study investigates the possible involvement of ECM components and signaling pathways in the regulation of resistance to anoikis in endothelial cells (EC). Endothelial cells submitted to stressful conditions by blocking adhesion to substrate (anoikis resistance) display an up-regulation of Ras/ERK and PI3k/Akt pathways by high expression of Ras, ERK, PI3K (p110α) and Akt (Thr 308). After ERK and PI3K inhibiting, all EC-derived cell lines studied showed lower growth, a decrease in invasive potential and a higher rate of apoptosis. Furthermore, anoikis-resistant cell lines display a decrease in the expression of fibronectin, collagen IV and hyaluronic acid and an increase in the expression of laminin, perlecan, αv, ß3, α5 and ß1 integrins subunits, hyaluronidades 1, 2 and 3 and metalloproteinases 2 and 9. These results indicate that the acquisition of anoikis resistance induced remodeling of the extracellular matrix and overexpression of the PI3K/Akt and Ras/ERK pathway components. Acquisition of resistance to anoikis is a potentially crucial step in endothelial cell transformation.

Concentration of glycosaminoglycan in ovariectomized mice uterus after treatment with ovarian steroids.

The aim of this study was to evaluate the amount of non- and sulfated glycosaminoglycans in the ovariectomized mice uterus, after treatment with ovarian steroids. For this purpose, 50 adult female mice were divided into five groups with 10 animals/each: control group: CG (ovary intact), and ovariectomized groups: OG (vehicle), EG (estradiol), PG (progesterone) and EPG (estradiol combined to progesterone). The treatments started 30 days after ovariectomy. All the animals were treated for 50 consecutive days. These hormones were administered in a sterile oily solution via gavage. Twenty-four hours after the last treatment, all animals were euthanized, removing the uterine horn for biochemical analyses. To quantify, the hyaluronic acid (HA) used ELISA-like fluorometric assay, and the sulfated glycosaminoglycans (GAGs) used agarose gel electrophoresis. The amount of HA was significantly higher in the group treated with progesterone (PG) compared to the others groups (p < 0.05), and in the group treated with estradiol (EG), the amount of chondroitin/dermatan sulfate was significantly higher compared to the others groups (p < 0.05), and in the group treated with progesterone (PG), the amount of heparan sulfate was significantly lower compared to the others groups, except to control group (p < 0.05). Our results showed that the estroprogestative therapy after long time (50 days) profoundly affected the amount of glycosaminoglycans in uterine. These changes may be indicative of uterine pathology such as the development of tumor.

Glycosaminoglycans affect heparanase location in CHO cell lines.

Glycosaminoglycans (GAG) play a ubiquitous role in tissues and cells. In eukaryotic cells, heparan sulfate (HS) is initially degraded by an endo-ß-glucuronidase called heparanase-1 (HPSE). HS oligosaccharides generated by the action of HPSE intensify the activity of signaling molecules, activating inflammatory response, tumor metastasis, and angiogenesis. The aim of the present study was to understand if sulfated GAG could modulate HPSE, since the mechanisms that regulate HPSE have not been completely defined. CHO-K1 cells were treated with 4-methylumbelliferone (4-MU) and sodium chlorate, to promote total inhibition of GAG synthesis, and reduce the sulfation pattern, respectively. The GAG profile of the wild CHO-K1 cells and CHO-745, deficient in xylosyltransferase, was determined after [(35)S]-sulfate labeling. HPSE expression was determined via real-time quantitative polymerase chain reaction. Total ablation of GAG with 4-MU in CHO-K1 inhibited HPSE expression, while the lack of sulfation had no effect. Interestingly, 4-MU had no effect in CHO-745 cells for these assays. In addition, a different enzyme location was observed in CHO-K1 wild-type cells, which presents HPSE mainly in the extracellular matrix, in comparison with the CHO-745 mutant cells, which is found in the cytoplasm. In view of our results, we can conclude that GAG are essential modulators of HPSE expression and location. Therefore, GAG profile could impact cell behavior mediated by the regulation of HPSE.

Altered hyaluronic acid content in tear fluid of patients with adenoviral conjunctivitis.

The adenoviral conjunctivitis is one of the biggest causes of conjunctival infection in the world. Conjunctivitis causes relatively nonspecific symptoms, as hyperaemia and chemosis. Even after biomicroscopy, complex laboratory tests, such as viral culture, are necessary to identify the pathogen or its etiology. To contribute to the better understanding of the pathobiology of the adenoviral conjunctivitis, the tear fluids of patients with unilateral acute adenovirus conjunctivitis (UAAC), normal donors (control) and patients with allergic conjunctivitis were analyzed. Tear samples were collected with Schirmer strips from control, allergic conjunctivitis and UAAC patients, diagnosed by clinical signs. UAAC tears were tested positive in viral cultures. After the elution, HA was quantified using an ELISA-like fluorometric assay and the protein profile was determined by SDS-PAGE. A profound increase in the HA tear content in UAAC patients was found when compared to control and ALC. This HA increase in UAAC tears remarkably was not observed in tears from contralateral eyes without clinical signs, nor in allergic conjunctivitis. In addition a distinct profile of UAAC tear proteins was observed in patients with UAAC. The quantification of HA in the tear fluid is a rapid, sensitive and specific test. This molecule might be a biomarker candidate for acute conjunctivitis.

Lumican expression, localization and antitumor activity in prostate cancer.

The stromal reaction surrounding tumors leads to the formation of a tumor-specific microenvironment, which may play either a restrictive role or a supportive role in the growth and progression of the tumors. Lumican, a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM), regulates collagen fibrillogenesis. Recently, lumican has also been shown to regulate cell behavior during embryonic development, tissue repair and tumor progression. The role of lumican in cancer varies according to the type of tumor. In this study we analyze the role of lumican in the pathogenesis of prostate cancer both in vivo and in vitro. Overall lumican up-regulation was observed in the primary tumors analyzed through both real-time PCR and immunostaining. The increase in lumican expression was observed in the reactive stroma surrounding prostate primary tumors with fibrotic deposition surrounding the acinar glands. In vitro analysis demonstrated that lumican inhibited both the migration and invasion of metastatic prostate cancer cells isolated from lymph node, bone and brain. Moreover, prostate cancer cells seeded on lumican presented a decrease in the formation of cellular projections, lamellipodia detected by a decreased rearrangement in ZO-1, keratin 8/18, integrin ß1 and MT1-MMP, and invadopodia detected by disruption of α-smooth muscle actin, cortactin and N-WASP. Moreover, a significant increase in prostate cancer cell invasion was observed through the peritoneum of lumican knockout mice, further demonstrating the restrictive role lumican present in the ECM has on prostate cancer invasion. In conclusion, lumican present in the reactive stroma surrounding prostate primary tumors plays a restrictive role on cancer progression, and we therefore postulate that lumican could be a valuable marker in prostate cancer staging.

Evaluation of the metabolism of glycosaminoglycans in patients with interstitial cystis.

INTRODUCTION: Painful bladder syndrome/interstitial cystitis (PBS/IC) pathogenesis is not fully known, but evidence shows that glycosaminoglycans (GAG) of bladder urothelium can participate in its genesis. The loss of these compounds facilitates the contact of urine compounds with deeper portions of bladder wall triggering an inflammatory process. We investigated GAG in urine and tissue of PBS/IC and pure stress urinary incontinence (SUI) patients to better understand its metabolism. MATERIALS AND METHODS: Tissue and urine of 11 patients with PBS/IC according to NIDDK criteria were compared to 11 SUI patients. Tissue samples were analyzed by histological, immunohistochemistry and immunofluorescence methods. Statistical analysis were performed using t Student test and Anova, considering significant when p < 0.05. RESULTS: PBS/IC patients had lower concentration of GAG in urine when compared to SUI (respectively 0.45 ± 0.11 x 0.62 ± 0.13 mg/mg creatinine, p < 0.05). However, there was no reduction of the content of GAG in the urothelium of both groups. Immunofluorescence showed that PBS/IC patients had a stronger staining of TGF-beta, decorin (a proteoglycan of chondroitin/dermatan sulfate), fibronectin and hyaluronic acid. CONCLUSION: the results suggest that GAG may be related to the ongoing process of inflammation and remodeling of the dysfunctional urothelium that is present in the PBS/IC.

Probing the interaction between heparan sulfate proteoglycan with biologically relevant molecules in mimetic models for cell membranes: a Langmuir film study.

Investigating the role of proteoglycans associated to cell membranes is fundamental to comprehend biochemical process that occurs at the level of membrane surfaces. In this paper, we exploit syndecan-4, a heparan sulfate proteoglycan obtained from cell cultures, in lipid Langmuir monolayers at the air-water interface. The monolayer served as a model for half a membrane, and the molecular interactions involved could be evaluated with tensiometry and vibrational spectroscopy techniques. Polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) employed in a constant surface pressure regime showed that the main chemical groups for syndecan-4 were present at the air-water interface. Subsequent monolayer decompression and compression showed surface pressure-area isotherms with a large expansion for the lipid monolayers interacting with the cell culture reported to over-express syndecan-4, which was also an indication that the proteoglycan was inserted in the lipid monolayer. The introduction of biological molecules with affinity for syndecam-4, such as growth factors, which present a key role in biochemical process of cell signaling, changed the surface properties of the hybrid film, leading to a model, by which the growth factor binds to the sulfate groups present in the heparan sulfate chains. The polypeptide moiety of syndecan-4 responds to this interaction changing its conformation, which leads to lipid film relaxation and further monolayer condensation.

Antiangiogenic activity and direct antitumor effect from a sulfated polysaccharide isolated from seaweed.

Angiogenesis is a dynamic proliferation and differentiation process. It requires endothelial proliferation, migration and tube formation. In this context, endothelial cells are a preferred target for several studies and therapies. Anionic polysaccharides (SV1 and PSV1) from brown seaweed Sargassum vulgare were fractionated (SV1), purified (PSV1) and displayed with high total sugars, sulfate content and very low level of protein. The antiangiogenic efficacy of polysaccharides was examined in vivo in the chick chorioallantoic membrane (CAM) model by using fertilized eggs. Decreases in the density of the capillaries were assessed and scored. The results showed that SV1 and PSV1 have an inhibitory effect on angiogenesis. These results were also confirmed by the inhibition of tubulogenesis in rabbit aorta endothelial cell (RAEC) in matrigel. These compounds were assessed in an apoptosis assay (Annexin V-FITC/PI) and cell viability by MTT assay of RAEC. These polysaccharides did not affect the viability and did not have apoptotic or necrotic action. RAEC cell when incubated with SV1and PSV1 showed inhibition of VEGF secretion, observed when compounds were incubated at 25, 50 and 100 µg/µL. The VEGF secretion with the RAEC cell line for 24 h was more effective for PSV1 at 50 µg/µL (71.4%) than for SV1 at 100 µg/µL (75.9%). SV1 and PSV1 had an antiproliferative action (47%) against tumor cell line HeLa. Our results indicate that these sulfated polysaccharides have antiangiogenic and antitumor actions.

Inhibitory peptides of the sulfotransferase domain of the heparan sulfate enzyme, N-deacetylase-N-sulfotransferase-1.

N-Deacetylase-N-sulfotransferase 1 (Ndst1) catalyzes the initial modification of heparan sulfate and heparin during their biosynthesis by removal of acetyl groups from subsets of N-acetylglucosamine units and subsequent sulfation of the resulting free amino groups. In this study, we used a phage display library to select peptides that interact with Ndst1, with the aim of finding inhibitors of the enzyme. The phage library consisted of cyclic random 10-mer peptides expressed in the phage capsid protein pIII. Selection was based on the ability of engineered phage to bind to recombinant murine Ndst1 (mNdst1) and displacement with heparin. Peptides that were enriched through multiple cycles of binding and disassociation displayed two specific sequences, CRGWRGEKIGNC and CNMQALSMPVTC. Both peptides inhibited mNdst1 activity in vitro, however, by distinct mechanisms. The peptide CRGWRGEKIGNC presents a chemokine-like repeat motif (BXX, where B represents a basic amino acid and X is a noncharged amino acid) and binds to heparan sulfate, thus blocking the binding of substrate to the enzyme. The peptide NMQALSMPVT inhibits mNdst1 activity by direct interaction with the enzyme near the active site. The discovery of inhibitory peptides in this way suggests a method for developing peptide inhibitors of heparan sulfate biosynthesis.

A novel approach for the characterisation of proteoglycans and biosynthetic enzymes in a snail model.

Proteoglycans encompass a heterogeneous group of glycoconjugates where proteins are substituted with linear, highly negatively charged glycosaminoglycan chains. Sulphated glycosaminoglycans are ubiquitous to the animal kingdom of the Eukarya domain. Information on the distribution and characterisation of proteoglycans in invertebrate tissues is limited and restricted to a few species. By the use of multidimensional protein identification technology and immunohistochemistry, this study shows for the first time the presence and tissue localisation of different proteoglycans, such as perlecan, aggrecan, and heparan sulphate proteoglycan, amongst others, in organs of the gastropoda Achatina fulica. Through a proteomic analysis of Golgi proteins and immunohistochemistry of tissue sections, we detected the machinery involved in glycosaminoglycan biosynthesis, related to polymer formation (polymerases), as well as secondary modifications (sulphation and uronic acid epimerization). Therefore, this work not only identifies both the proteoglycan core proteins and glycosaminoglycan biosynthetic enzymes in invertebrates but also provides a novel method for the study of glycosaminoglycan and proteoglycan evolution.