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Receptor-mediated transcytosis (RMT) is a principal pathway for transport of macromolecules essential for brain function across the blood-brain barrier (BBB). Antibodies or peptide ligands which bind RMT receptors are often co-opted for brain delivery of biotherapeutics. Constitutively recycling transferrin receptor (TfR) is a prototype receptor utilized to shuttle therapeutic cargos across the BBB. Several other BBB-expressed receptors have been shown to mediate transcytosis of antibodies or protein ligands including insulin receptor (INSR) and insulin-like growth factor-1 receptor (IGF1R), lipid transporters LRP1, LDLR, LRP8 and TMEM30A, solute carrier family transporter SLC3A2/CD98hc and leptin receptor (LEPR). In this study, we analyzed expression patterns of genes encoding RMT receptors in isolated brain microvessels, brain parenchyma and peripheral organs of the mouse and the human using RNA-seq approach. IGF1R, INSR and LRP8 were highly enriched in mouse brain microvessels compared to peripheral tissues. In human brain microvessels only INSR was enriched compared to either the brain or the lung. The expression levels of SLC2A1, LRP1, IGF1R, LRP8 and TFRC were significantly higher in the mouse compared to human brain microvessels. The protein expression of these receptors analyzed by Western blot and immunofluorescent staining of the brain microvessels correlated with their transcript abundance. This study provides a molecular transcriptomics map of key RMT receptors in mouse and human brain microvessels and peripheral tissues, important to translational studies of biodistribution, efficacy and safety of antibodies developed against these receptors.

Activity screening and insertional inactivation of lipopolysaccharide (LPS) biosynthetic genes in Helicobacter pylori have led to the successful characterization of two key enzymes encoded by HP0159 (JHP0147) and HP1105 (JHP1032) open reading frames (ORFs) which are members of the large and diverse carbohydrate active enzymes (CAZY) GT-8 (rfaJ) family of glycosyltransferases. Activity screening of a genomic library led to the identification of the enzyme involved in the biosynthesis of the type 2 N-acetyl-lactosamine O-chain backbone, the beta-1,3-N-acetyl-glucosaminyl transferase. In addition, the activity screening approach led to the identification and characterization of a key core biosynthetic enzyme responsible for the biosynthesis of the alpha-1,6-glucan polymer. This alpha-1,6-glucosyltransferase protein is encoded by the HP0159 ORF. Both enzymes play an integral part in the biosynthesis of LPS, and insertional inactivation leads to the production of a truncated LPS molecule on the bacterial cell surface. The LPS structures were determined by mass spectrometry and chemical analyses. The linkage specificity of each glycosyltransferase was determined by nuclear magnetic resonance (NMR) analysis of model compounds synthesized in vitro. A cryogenic probe was used to structurally characterize nanomole amounts of the product of the HP1105 (JHP1032) enzyme. In contrast to the HP0159 enzyme, which displays the GT-8-predicted retaining stereochemistry for the reaction product, HP1105 (JHP1032) is the first member of this GT-8 family to have been shown to have an inverting stereochemistry in its reaction products.

In addition to sialic acid, bacteria produce several other nonulosonic acids, including legionaminic acid (Leg). This has exactly the same stereochemistry as sialic acid, with the added features of 9-deoxy and 7-amino groups. In order to explore the biological effects of replacing sialic acid residues (Neu5Ac) in glycoconjugates with Leg in its diacetylated form, diacetyllegionaminic acid (Leg5Ac7Ac), we tested CMP-Leg5Ac7Ac as a donor substrate with a selection of bacterial and mammalian sialyltransferases. The CMP-Leg5Ac7Ac was synthesized in vitro by means of cloned enzymes from the bacillosamine portion of the Campylobacter jejuni N-glycan pathway and from the Leg pathway of Legionella pneumophila. Using fluorescent derivatives of lactose, Galβ1,4GlcNAcβ and T-antigen (Galβ1,3GalNAcα) as acceptors, we tested eight different sialyltransferases and found that the Pasteurella multocida PM0188h and porcine ST3Gal1 sialyltransferases were significantly active with CMP-Leg5Ac7Ac, showing ∼60% activity when compared with CMP-Neu5Ac. The Photobacterium α2,6 sialyltransferase was weakly active, with ∼6% relative activity. The Leg5Ac7Ac-α-2,3-lactose product was then tested as a substrate with six sialidases of viral, bacterial and mammalian origin. All showed much lower activities than with the corresponding sialic acid substrate, with the influenza virus N1 being the most active and human NEU2 being the least active. These results show the feasibility of producing glycoconjugates with Leg5Ac7Ac residues as the terminal sugars, which should display novel biological properties.

Legionaminic acids (Leg) are bacterial analogs of neuraminic acid, with the same stereochemistry but different substituents at C5, C7 and C9. Hence they may be incorporated into useful analogs of sialoglycoconjugates, and we previously reported two sialyltransferases that could utilize cytidine monophosphate (CMP)-Leg5Ac7Ac for preparation of Leg glycoconjugates, which were resistant to sialidases [Watson DC, Leclerc S, Wakarchuk WW, Young NM. 2011. Enzymatic synthesis and properties of glycoconjugates with legionaminic acid as a replacement for neuraminic acid. Glycobiology. 21:99-108.]. These were the porcine ST3Gal1 and Pasteurella multocida sialyltransferases. We now report two additional sialyltransferases with superior Leg-transferase properties to the previous two. These are (i) a truncated form of a Photobacterium α2,6-sialyltransferase with an Ala-Met mutation in its active site, and (ii) an α2,3-sialyltransferase from Neisseria meningitidis MC58 with a higher transferase activity than the P. multocida enzyme, with either CMP-Neu5Ac or CMP-Leg5Ac7Ac as the donor. These enzymes will enable the production of useful Leg5Ac7Ac glycoconjugate derivatives with either α2,6 or α2,3 linkages and unique biological properties.