Loss of hepatic manganese transporter ZIP8 disrupts serum transferrin glycosylation and the glutamate-glutamine cycle.

BACKGROUND: ZIP8, encoded by SLC39A8, is a membrane transporter that facilitates the cellular uptake of divalent biometals including zinc (Zn), manganese (Mn), and iron (Fe). The hepatic system has long been accepted as the central modulator for whole-body biometal distribution. Earlier investigations suggest the propensity of ZIP8 to prioritize Mn influx, as opposed to Fe or Zn, in hepatocytes. Hepatic ZIP8 Mn transport is crucial for maintaining homeostasis of various Mn-dependent metalloenzymes and their associated pathways. Herein, we hypothesize that a drastic decrease in systemic Mn, via the loss of hepatic ZIP8, disrupts two unique cellular pathways, post-translational glycosylation and the glutamate-glutamine cycle. METHODS: ZIP8 liver-specific knockout (LSKO) mice were chosen in an attempt to substantially decrease whole-body Mn levels. To further elucidate the role of Mn in serum glycosylation, a Mn-deficient diet was adopted in conjunction with the LSKO mice to model a near-complete loss of systemic Mn. After the treatment course, transferrin sialylation profiles were determined using imaged capillary isoelectric focusing (icIEF). We also investigated the role of Mn in the glutamate-glutamine cycle; the conversion of glutamate to glutamine in F/F and LSKO mice was assessed by the glutamine/glutamate ratio in cerebrospinal fluid (CSF) via HPLC-MS. An open-field study was ultimately conducted to check if these mice displayed atypical behavior. RESULTS: Two major biological pathways were found to be significantly altered due to the loss of hepatic ZIP8. We identified a disparity between F/F and LSKO transferrin sialylation profiles that were exacerbated under a Mn-deficient diet. Additionally, we discovered a neurotransmitter imbalance between the levels of glutamine and glutamate, exclusive to LSKO mice. This was characterized by the decreased glutamine/glutamate ratio in CSF. Secondary to the neurotransmitter alteration, LSKO mice exhibited an increase in locomotor activity in an open-field. CONCLUSION: Our model successfully established a connection between the loss of hepatic ZIP8 and two Mn-dependent cellular pathways, namely, protein glycosylation and the glutamate-glutamine cycle.

Development of a highly sensitive imaged cIEF immunoassay for studying AAV capsid protein charge heterogeneity.

Post-translational modifications (PTMs) of adeno-associated virus (AAV) capsid proteins tune and regulate the AAV infective life cycle, which can impact the safety and efficacy of AAV gene therapy products. Many of these PTMs induce changes in protein charge heterogeneity, including deamidation, oxidation, glycation, and glycosylation. To characterize the charge heterogeneity of a protein, imaged capillary isoelectric focusing (icIEF) has become the gold standard method. We have previously reported an icIEF method with native fluorescence detection for denatured AAV capsid protein charge heterogeneity analysis. Although well suited for final products, the method does not have sufficient sensitivity for upstream, low-concentration AAV samples, and lacks the specificity for capsid protein detection in complex samples like cell culture supernatants and cell lysates. In contrast, the combination of icIEF, protein capture, and immunodetection affords significantly higher sensitivity and specificity, addressing the challenges of the icIEF method. By leveraging different primary antibodies, the icIEF immunoassay provides additional selectivity and affords a detailed characterization of individual AAV capsid proteins. In this study, we describe an icIEF immunoassay method for AAV analysis that is 90 times more sensitive than native fluorescence icIEF. This icIEF immunoassay provides AAV stability monitoring, where changes in individual capsid protein charge heterogeneity can be observed in response to heat stress. When applied to different AAV serotypes, this method also provides serotype identity with reproducible quantification of VP protein peak areas and apparent isoelectric point (pI). Overall, the described icIEF immunoassay is a sensitive, reproducible, quantitative, specific, and selective tool that can be used across the AAV biomanufacturing process, especially in upstream process development where complex sample types are often encountered.