Opposing roles for ADAMTS2 and ADAMTS14 in myofibroblast differentiation and function.

Crosstalk between cancer and stellate cells is pivotal in pancreatic cancer, resulting in differentiation of stellate cells into myofibroblasts that drives tumour progression. To assess cooperative mechanisms in a 3D context, we generated chimeric spheroids using human and mouse cancer and stellate cells. Species-specific deconvolution of bulk-RNA sequencing data revealed cell type-specific transcriptomes underpinning invasion. This dataset highlighted stellate-specific expression of transcripts encoding the collagen-processing enzymes ADAMTS2 and ADAMTS14. Strikingly, loss of ADAMTS2 reduced, while loss of ADAMTS14 promoted, myofibroblast differentiation and invasion independently of their primary role in collagen-processing. Functional and proteomic analysis demonstrated that these two enzymes regulate myofibroblast differentiation through opposing roles in the regulation of transforming growth factor ß availability, acting on the protease-specific substrates, Serpin E2 and fibulin 2, for ADAMTS2 and ADAMTS14, respectively. Showcasing a broader complexity for these enzymes, we uncovered a novel regulatory axis governing malignant behaviour of the pancreatic cancer stroma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Basement membrane stiffness determines metastases formation.

The basement membrane (BM) is a special type of extracellular matrix and presents the major barrier cancer cells have to overcome multiple times to form metastases. Here we show that BM stiffness is a major determinant of metastases formation in several tissues and identify netrin-4 (Net4) as a key regulator of BM stiffness. Mechanistically, our biophysical and functional analyses in combination with mathematical simulations show that Net4 softens the mechanical properties of native BMs by opening laminin node complexes, decreasing cancer cell potential to transmigrate this barrier despite creating bigger pores. Our results therefore reveal that BM stiffness is dominant over pore size, and that the mechanical properties of 'normal' BMs determine metastases formation and patient survival independent of cancer-mediated alterations. Thus, identifying individual Net4 protein levels within native BMs in major metastatic organs may have the potential to define patient survival even before tumour formation. The ratio of Net4 to laminin molecules determines BM stiffness, such that the more Net4, the softer the BM, thereby decreasing cancer cell invasion activity.

Predictive Factors for Revision and Survivorship Analysis of a Prevalent 36-mm Metal-on-Metal Total Hip Replacement System: A Large Single-Center Retrospective Cohort Study.

BACKGROUND: To our knowledge, this is the largest single-center cohort of the 36-mm Corail-Pinnacle metal-on-metal total hip replacements system, aiming to determine 10-year survivorship and identify predictors of revision. We further assessed year of implantation given reports of manufacturing variations affecting shells made after 2006 predisposing these components to increasing wear. METHODS: All Corail-Pinnacle 36-mm metal-on-metal hips implanted in a single center (2005-2012). The effect of patient and implant-related variables, and year of implantation on revision risk was assessed using Kaplan-Meier, Cox regression, and interrupted time series analysis. RESULTS: In total, 1212 metal-on-metal total hip replacements were implanted with a 10-year survival rate of 83.4% (95% confidence interval [CI] = 81.3-85.5). Mean follow-up duration was 7.3 years with 61% of patients reaching a minimum of 7 years of follow-up. One hundred nineteen patients required revision surgery (9.8%). Univariate analysis identified female gender (hazard ratio [HR] = 1.608, CI = 1.093-2.364, P = .016), age at implantation (HR = 0.982, CI = 0.968-0.997, P = .019), smaller 50-mm to 54-mm cup diameter (HR = 1.527, CI = 1.026-2.274, P = .037), and high-offset stems (HR = 2.573, CI = 1.619-4.089, P < .001) as predictors of revision. Multivariate modeling confirmed female gender and high-offset stems as significant predictors of revision. For components implanted after 2007, the number of revisions showed no statistically significant step increase compared to pre-2007 implantation. CONCLUSION: We observed a high 10-year failure rate (16.6%) with this implant, mostly due to adverse reaction to metal debris. Female gender and high femoral offset stems were significant predictors for all-cause revision. Year of implantation was not significantly associated with an increasing number of revisions from 2007 onwards, although further studies to validate the impact of manufacturing discrepancies are recommended.

N-glycolyl groups of nonhuman chondroitin sulfates survive in ancient fossils.

Biosynthesis of the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) was lost during human evolution due to inactivation of the CMAH gene, possibly expediting divergence of the Homo lineage, due to a partial fertility barrier. Neu5Gc catabolism generates N-glycolylhexosamines, which are potential precursors for glycoconjugate biosynthesis. We carried out metabolic labeling experiments and studies of mice with human-like Neu5Gc deficiency to show that Neu5Gc degradation is the metabolic source of UDP-GlcNGc and UDP-GalNGc and the latter allows an unexpectedly selective incorporation of N-glycolyl groups into chondroitin sulfate (CS) over other potential glycoconjugate products. Partially N-glycolylated-CS was chemically synthesized as a standard for mass spectrometry to confirm its natural occurrence. Much lower amounts of GalNGc in human CS can apparently be derived from Neu5Gc-containing foods, a finding confirmed by feeding Neu5Gc-rich chow to human-like Neu5Gc-deficient mice. Unlike the case with Neu5Gc, N-glycolyl-CS was also stable enough to be detectable in animal fossils as old as 4 My. This work opens the door for investigating the biological and immunological significance of this glycosaminoglycan modification and for an "ancient glycans" approach to dating of Neu5Gc loss during the evolution of Homo.

Cancer glycan epitopes: biosynthesis, structure and function.

Aberrant glycan epitopes are a classic hallmark of malignant transformation, yet their full clinical potential in cancer diagnostics and therapeutics is yet to be realized. This is partly because our understanding of how these epitopes are regulated remains poorly understood. In this review cancer glycan epitopes for the major glycan classes are summarized with a focus on their biosynthesis, structure and role in cancer progression. Their application as cancer biomarkers, in particular the more recent work on cancer glycoforms, and the advantages these offer over the glycan or protein alone are discussed. Finally, emerging concepts which expand on the current view of the cancer glycan epitope beyond the single structure, to patterns and the whole glycocalyx, are described. These new approaches that consider the cancer glycan epitope as a glycoform, or as a pattern of many epitope structures, are providing new targets both for cancer biomarkers and therapeutics currently in development at the bench and the clinic.

A red meat-derived glycan promotes inflammation and cancer progression.

A well known, epidemiologically reproducible risk factor for human carcinomas is the long-term consumption of "red meat" of mammalian origin. Although multiple theories have attempted to explain this human-specific association, none have been conclusively proven. We used an improved method to survey common foods for free and glycosidically bound forms of the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc), showing that it is highly and selectively enriched in red meat. The bound form of Neu5Gc is bioavailable, undergoing metabolic incorporation into human tissues, despite being a foreign antigen. Interactions of this antigen with circulating anti-Neu5Gc antibodies could potentially incite inflammation. Indeed, when human-like Neu5Gc-deficient mice were fed bioavailable Neu5Gc and challenged with anti-Neu5Gc antibodies, they developed evidence of systemic inflammation. Such mice are already prone to develop occasional tumors of the liver, an organ that can incorporate dietary Neu5Gc. Neu5Gc-deficient mice immunized against Neu5Gc and fed bioavailable Neu5Gc developed a much higher incidence of hepatocellular carcinomas, with evidence of Neu5Gc accumulation. Taken together, our data provide an unusual mechanistic explanation for the epidemiological association between red meat consumption and carcinoma risk. This mechanism might also contribute to other chronic inflammatory processes epidemiologically associated with red meat consumption.

Characterization of the Extracellular Matrix of Normal and Diseased Tissues Using Proteomics.

The extracellular matrix (ECM) is a complex meshwork of insoluble fibrillar proteins and signaling factors interacting together to provide architectural and instructional cues to the surrounding cells. Alterations in ECM organization or composition and excessive ECM deposition have been observed in diseases such as fibrosis, cardiovascular diseases, and cancer. We provide here optimized protocols to solubilize ECM proteins from normal or tumor tissues, digest the proteins into peptides, analyze ECM peptides by mass spectrometry, and interpret the mass spectrometric data. In addition, we present here two novel R-script-based web tools allowing rapid annotation and relative quantification of ECM proteins, peptides, and intensity/abundance in mass spectrometric data output files. We illustrate this protocol with ECMs obtained from two pairs of tissues, which differ in ECM content and cellularity: triple-negative breast cancer and adjacent mammary tissue, and omental metastasis from high-grade serous ovarian cancer and normal omentum. The complete proteomics data set generated in this study has been deposited to the public repository ProteomeXchange with the data set identifier: PXD005554.

Inverse hormesis of cancer growth mediated by narrow ranges of tumor-directed antibodies.

Compelling evidence for naturally occurring immunosurveillance against malignancies informs and justifies some current approaches toward cancer immunotherapy. However, some types of immune reactions have also been shown to facilitate tumor progression. For example, our previous studies showed that although experimental tumor growth is enhanced by low levels of circulating antibodies directed against the nonhuman sialic acid N-glycolyl-neuraminic acid (Neu5Gc), which accumulates in human tumors, growth could be inhibited by anti-Neu5Gc antibodies from a different source, in a different model. However, it remains generally unclear whether the immune responses that mediate cancer immunosurveillance vs. those responsible for inflammatory facilitation are qualitatively and/or quantitatively distinct. Here, we address this question using multiple murine tumor growth models in which polyclonal antibodies against tumor antigens, such as Neu5Gc, can alter tumor progression. We found that although growth was stimulated at low antibody doses, it was inhibited by high doses, over a linear and remarkably narrow range, defining an immune response curve (IRC; i.e., inverse hormesis). Moreover, modulation of immune responses against the tumor by altering antibody avidity or by enhancing innate immunity shifted the IRC in the appropriate direction. Thus, the dualistic role of immunosurveillance vs. inflammation in modulating tumor progression can be quantitatively distinguished in multiple model systems, and can occur over a remarkably narrow range. Similar findings were made in a human tumor xenograft model using a narrow range of doses of a monoclonal antibody currently in clinical use. These findings may have implications for the etiology, prevention, and treatment of cancer.

Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer.

Certain pathogenic bacteria are known to modulate the innate immune response by decorating themselves with sialic acids, which can engage the myelomonocytic lineage inhibitory receptor Siglec-9, thereby evading immunosurveillance. We hypothesized that the well-known up-regulation of sialoglycoconjugates by tumors might similarly modulate interactions with innate immune cells. Supporting this hypothesis, Siglec-9-expressing myelomonocytic cells found in human tumor samples were accompanied by a strong up-regulation of Siglec-9 ligands. Blockade of Siglec-9 enhanced neutrophil activity against tumor cells in vitro. To investigate the function of inhibitory myelomonocytic Siglecs in vivo we studied mouse Siglec-E, the murine functional equivalent of Siglec-9. Siglec-E-deficient mice showed increased in vivo killing of tumor cells, and this effect was reversed by transgenic Siglec-9 expression in myelomonocytic cells. Siglec-E-deficient mice also showed enhanced immunosurveillance of autologous tumors. However, once tumors were established, they grew faster in Siglec-E-deficient mice. In keeping with this, Siglec-E-deficient macrophages showed a propensity toward a tumor-promoting M2 polarization, indicating a secondary role of CD33-related Siglecs in limiting cancer-promoting inflammation and tumor growth. Thus, we define a previously unidentified impact of inhibitory myelomonocytic Siglecs in cancer biology, with distinct roles that reflect the dual function of myelomonocytic cells in cancer progression. In keeping with this, a human polymorphism that reduced Siglec-9 binding to carcinomas was associated with improved early survival in non-small-cell lung cancer patients, which suggests that Siglec-9 might be therapeutically targeted within the right time frame and stage of disease.

Rapid Trimming of Cell Surface Polysialic Acid (PolySia) by Exovesicular Sialidase Triggers Release of Preexisting Surface Neurotrophin.

As acidic glycocalyx on primary mouse microglial cells and a mouse microglial cell line Ra2, expression of polysialic acid (polySia/PSA), a polymer of the sialic acid Neu5Ac (N-acetylneuraminic acid), was demonstrated. PolySia is known to modulate cell adhesion, migration, and localization of neurotrophins mainly on neural cells. PolySia on Ra2 cells disappeared very rapidly after an inflammatory stimulus. Results of knockdown and inhibitor studies indicated that rapid surface clearance of polySia was achieved by secretion of endogenous sialidase Neu1 as an exovesicular component. Neu1-mediated polySia turnover was accompanied by the release of brain-derived neurotrophic factor normally retained by polySia molecules. Introduction of a single oxygen atom change into polySia by exogenous feeding of the non-neural sialic acid Neu5Gc (N-glycolylneuraminic acid) caused resistance to Neu1-induced polySia turnover and also inhibited the associated release of brain-derived neurotrophic factor. These results indicate the importance of rapid turnover of the polySia glycocalyx by exovesicular sialidases in neurotrophin regulation.

Sialic acids in cancer biology and immunity.

During malignant transformation, glycosylation is heavily altered compared with healthy tissue due to differential expression of glycosyltransferases, glycosidases and monosaccharide transporters within the cancer microenvironment. One key change of malignant tissue glycosylation is the alteration of sialic acid processing that leads to a general upregulation of sialylated glycans (hypersialylation) on cell surfaces and an increased introduction of the non-human sialic acid N-glycolyl-neuraminic acid (Neu5Gc) instead of N-acetyl-neuraminic acid into cell surface glycans. These changes have been shown to be the result of altered sialyltransferase and sialidase expression. Functionally, cancer-associated hypersialylation appears to directly impact tumor cell interaction with the microenvironment, in particular the modulation of sialic acid-binding lectins on immune cells. Moreover, Neu5Gc expression in human tissues enhances inflammation due to an anti-Neu5Gc immune response, which can potentially influence inflammation-induced cancer and cancer-associated inflammation. In this review, we summarize the changes of sialic acid biology within the malignant microenvironment and the resulting effect on cancer immunity.

Enhanced T cell function in a mouse model of human glycosylation.

Clinical evidence for a more active immune response in humans compared with our closest hominid relative, the chimpanzee, includes the progression of HIV infection to AIDS, hepatitis B- and C-related inflammation, autoimmunity, and unwanted harmful immune responses to viral gene transfer vectors. Humans have a unique mutation of the enzyme CMP-N-acetylneuraminic acid hydroxylase (CMAH), causing loss of expression of the sialic acid Neu5Gc. This mutation, occurring 2 million years ago, likely altered the expression and function of ITIM-bearing inhibitory receptors (Siglecs) that bind sialic acids. Previous work showed that human T cells proliferate faster than chimpanzee T cells upon equivalent stimulation. In this article, we report that Cmah(-/-) mouse T cells proliferate faster and have greater expression of activation markers than wild-type mouse T cells. Metabolically reintroducing Neu5Gc diminishes the proliferation and activation of both human and murine Cmah(-/-) T cells. Importantly, Cmah(-/-) mice mount greater T cell responses to an adenovirus encoding an adeno-associated virus capsid transgene. Upon lymphocytic choriomeningitis virus infection, Cmah(-/-) mice make more lymphocytic choriomeningitis virus-specific T cells than WT mice, and these T cells are more polyfunctional. Therefore, a uniquely human glycosylation mutation, modeled in mice, leads to a more proliferative and active T cell population. These findings in a human-like mouse model have implications for understanding the hyperimmune responses that characterize some human diseases.

Anatomic Reversal of Shoulder Joint After Neck of Humerus Fracture.

We report on a singular case of a unique form of post-traumatic reversal of the humeral head after humeral neck fracture, in which the pattern of collapse resulted in the formation of a native reverse polarity shoulder. In essence, the humeral head became a socket, and the glenoid rounded to become a head with well-preserved shoulder function. To our knowledge, this is the first case of an acquired shoulder deformity that bears a remarkable functional similarity to a prosthetic reverse polarity shoulder replacement.

Hip Dysplasia in a Patient in Late Adolescence With Charcot-Marie-Tooth and Multiple Acyl-CoA Dehydrogenase Deficiency.

This case report explores a unique presentation of hip dysplasia in a female patient aged 21 years old diagnosed with Charcot-Marie-Tooth disease (CMT) type 1A and multiple acyl-CoA dehydrogenase deficiency (MADD). The coexistence of these neuromuscular and metabolic disorders in a patient with hip dysplasia provides an opportunity to investigate their potential interactions and impact on diagnosis, treatment, and prognosis. The patient underwent labral repair with shelf osteotomy and later a total hip replacement. This case highlights the need for further research to better understand the relationships between CMT, MADD, neuromuscular dysplasia, and hip dysplasia. A deeper understanding of these interactions may lead to improved diagnostic techniques, earlier intervention, and personalized treatment approaches for patients with co-morbid conditions, ultimately improving patient outcomes and reducing complications later in life.

Orthogeriatric Care Following Hip Fracture: Improving Post-Operative Outcomes in an Aged Population.

INTRODUCTION: Hip fractures globally are associated with high levels of morbidity, mortality, and significant financial burden. This audit aimed to assess the impact of orthogeriatric liaison care on post-operative outcomes following surgical management of neck or femur fractures. METHODS: Here, 258 patients who underwent hip fracture surgery over 1-year were included. Data were collected as an audit following the transition to an orthogeriatric liaison care model, involving regular orthogeriatric review (thrice weekly ward rounds, daily board rounds), superseding orthogeriatric review as requested. The audit is meant to assess the development of post-operative non-surgical site infection (NSSI) and mortality and duration of inpatient stay. Outcomes were compared to previous data from our hospital site in 2015/2016. RESULTS: Patients with severe cognitive impairment and systemic disease (Abbreviated Mental Test Score (AMTS) < 7 and American Society of Anesthesiologists (ASA) grade ≥ 3) showed significantly elevated NSSI risk, consistent across the study periods. Both periods demonstrated an increased risk of NSSI associated with admission from nursing homes. Despite the 2021/2022 cohort being notably older, NSSI risk decreased from 40.6% to 37.2% after implementing the orthogeriatric care model. NSSI risk was notably reduced for severe cognitive impairment (51.6% vs. 71%), and the p-value was 0.025. Average hospital stay decreased post-intervention (2.4 days shorter), with a notable reduction for NSSI patients (3.4 days shorter). Overall mortality rates were similar, although mortality due to infection was significantly reduced in 2021/2022 (44.4% vs. 93.3%), and the p-value was 0.003. CONCLUSION: The orthogeriatric liaison care model significantly decreased NSSI only in individuals with severe cognitive impairment and infection-associated mortality. This highlights the integral role of orthogeriatricians in the care of elderly hip fracture patients.

A Novel Primary Cilium-Mediated Mechanism Through which Osteocytes Regulate Metastatic Behavior of Both Breast and Prostate Cancer Cells.

Bone metastases are a common cause of suffering in breast and prostate cancer patients, however, the interaction between bone cells and cancer cells is poorly understood. Using a series of co-culture, conditioned media, human cancer spheroid, and organ-on-a-chip experiments, this study reveals that osteocytes suppress cancer cell proliferation and increase migration via tumor necrosis factor alpha (TNF-α) secretion. This action is regulated by osteocyte primary cilia and associated intraflagellar transport protein 88 (IFT88). Furthermore, it shows that cancer cells block this mechanism by secreting transforming growth factor beta (TGF-ß), which disrupts osteocyte cilia and IFT88 gene expression. This bi-directional crosstalk signaling between osteocytes and cancer cells is common to both breast and prostate cancer. This study also proposes that osteocyte inhibition of cancer cell proliferation decreases as cancer cells increase, producing more TGF-ß. Hence, a positive feedback loop develops accelerating metastatic tumor growth. These findings demonstrate the importance of cancer cell-osteocyte signaling in regulating breast and prostate bone metastases and support the development of therapies targeting this pathway.

Versican Associates with Tumor Immune Phenotype and Limits T-cell Trafficking via Chondroitin Sulfate.

Immunotherapies for cancers of epithelial origin have limited efficacy, and a growing body of evidence links the composition of extracellular matrix (ECM) with the likelihood of a favorable response to treatment. The ECM may be considered an immunologic barrier, restricting the localization of cytotoxic immune cells to stromal areas and inhibiting their contact with tumor cells. Identifying ECM components of this immunologic barrier could provide targets that whether degraded in situ may support antitumor immunity and improve immunotherapy response. Using a library of primary triple-negative breast cancer tissues, we correlated CD8+ T-cell tumor contact with ECM composition and identified a proteoglycan, versican (VCAN), as a putative member of the immunologic barrier. Our analysis reveals that CD8+ T-cell contact with tumor associates with the location of VCAN expression, the specific glycovariant of VCAN [defined through the pattern of posttranslational attachments of glycosaminoglycans (GAG)], and the cell types that produce the variant. In functional studies, the isomers of chondroitin sulfate presented on VCAN have opposing roles being either supportive or inhibiting of T-cell trafficking, and removal of the GAGs ameliorates these effects on T-cell trafficking. Overall, we conclude that VCAN can either support or inhibit T-cell trafficking within the tumor microenvironment depending on the pattern of GAGs present, and that VCAN is a major component of the ECM immunologic barrier that defines the type of response to immunotherapy. SIGNIFICANCE: The response to immunotherapy has been poor toward solid tumors despite immune cells infiltrating into the tumor. The ECM has been associated with impacting T-cell infiltration toward the tumor and in this article we have identified VCAN and its structural modification, chondroitin sulfate as having a key role in T-cell invasion.

A Self-Assembled 3D Model Demonstrates How Stiffness Educates Tumor Cell Phenotypes and Therapy Resistance in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense and stiff extracellular matrix (ECM) associated with tumor progression and therapy resistance. To further the understanding of how stiffening of the tumor microenvironment (TME) contributes to aggressiveness, a three-dimensional (3D) self-assembling hydrogel disease model is developed based on peptide amphiphiles (PAs, PA-E3Y) designed to tailor stiffness. The model displays nanofibrous architectures reminiscent of native TME and enables the study of the invasive behavior of PDAC cells. Enhanced tuneability of stiffness is demonstrated by interacting thermally annealed aqueous solutions of PA-E3Y (PA-E3Yh) with divalent cations to create hydrogels with mechanical properties and ultrastructure similar to native tumor ECM. It is shown that stiffening of PA-E3Yh hydrogels to levels found in PDAC induces ECM deposition, promotes epithelial-to-mesenchymal transition (EMT), enriches CD133+/CXCR4+ cancer stem cells (CSCs), and subsequently enhances drug resistance. The findings reveal how a stiff 3D environment renders PDAC cells more aggressive and therefore more faithfully recapitulates in vivo tumors.

Metabolism of vertebrate amino sugars with N-glycolyl groups: elucidating the intracellular fate of the non-human sialic acid N-glycolylneuraminic acid.

The two major mammalian sialic acids are N-acetylneuraminic acid and N-glycolylneuraminic acid (Neu5Gc). The only known biosynthetic pathway generating Neu5Gc is the conversion of CMP-N-acetylneuraminic acid into CMP-Neu5Gc, which is catalyzed by the CMP-Neu5Ac hydroxylase enzyme. Given the irreversible nature of this reaction, there must be pathways for elimination or degradation of Neu5Gc, which would allow animal cells to adjust Neu5Gc levels to their needs. Although humans are incapable of synthesizing Neu5Gc due to an inactivated CMAH gene, exogenous Neu5Gc from dietary sources can be metabolically incorporated into tissues in the face of an anti-Neu5Gc antibody response. However, the metabolic turnover of Neu5Gc, which apparently prevents human cells from continued accumulation of this immunoreactive sialic acid, has not yet been elucidated. In this study, we show that pre-loaded Neu5Gc is eliminated from human cells over time, and we propose a conceivable Neu5Gc-degrading pathway based on the well studied metabolism of N-acetylhexosamines. We demonstrate that murine tissue cytosolic extracts harbor the enzymatic machinery to sequentially convert Neu5Gc into N-glycolylmannosamine, N-glycolylglucosamine, and N-glycolylglucosamine 6-phosphate, whereupon irreversible de-N-glycolylation of the latter results in the ubiquitous metabolites glycolate and glucosamine 6-phosphate. We substantiate this finding by demonstrating activity of recombinant human enzymes in vitro and by studying the fate of radiolabeled pathway intermediates in cultured human cells, suggesting that this pathway likely occurs in vivo. Finally, we demonstrate that the proposed degradative pathway is partially reversible, showing that N-glycolylmannosamine and N-glycolylglucosamine (but not glycolate) can serve as precursors for biosynthesis of endogenous Neu5Gc.

Metabolism of vertebrate amino sugars with N-glycolyl groups: incorporation of N-glycolylhexosamines into mammalian glycans by feeding N-glycolylgalactosamine.

The outermost positions of mammalian cell-surface glycans are predominantly occupied by the sialic acids N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). To date, hydroxylation of CMP-Neu5Ac resulting in the conversion into CMP-Neu5Gc is the only known enzymatic reaction in mammals to synthesize a monosaccharide carrying an N-glycolyl group. In our accompanying paper (Bergfeld, A. K., Pearce, O. M., Diaz, S. L., Pham, T., and Varki, A. (2012) J. Biol. Chem. 287, jbc.M112.363549), we report a metabolic pathway for degradation of Neu5Gc, demonstrating that N-acetylhexosamine pathways are tolerant toward the N-glycolyl substituent of Neu5Gc breakdown products. In this study, we show that exogenously added N-glycolylgalactosamine (GalNGc) serves as a precursor for Neu5Gc de novo biosynthesis, potentially involving seven distinct mammalian enzymes. Following the GalNAc salvage pathway, UDP-GalNGc is epimerized to UDP-GlcNGc, which might compete with the endogenous UDP-GlcNAc for the sialic acid biosynthetic pathway. Using UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase-deficient cells, we confirm that conversion of GalNGc into Neu5Gc depends on this key enzyme of sialic acid biosynthesis. Furthermore, we demonstrate by mass spectrometry that the metabolic intermediates UDP-GalNGc and UDP-GlcNGc serve as substrates for assembly of most major classes of cellular glycans. We show for the first time incorporation of GalNGc and GlcNGc into chondroitin/dermatan sulfates and heparan sulfates, respectively. As demonstrated by structural analysis, N-glycolylated hexosamines were found in cellular gangliosides and incorporated into Chinese hamster ovary cell O-glycans. Remarkably, GalNAc derivatives altered the overall O-glycosylation pattern as indicated by the occurrence of novel O-glycan structures. This study demonstrates that mammalian N-acetylhexosamine pathways and glycan assembly are surprisingly tolerant toward the N-glycolyl substituent.