Cell type-dependent variations in the subcellular distribution of alpha-mannosidase I and II

alpha-mannosidases I and II (Man I and II) are resident enzymes of the Golgi complex involved in oligosaccharide processing during N-linked glycoprotein biosynthesis that are widely considered to be markers of the cis- and medial-Golgi compartments, respectively. We have investigated the distribution of these enzymes in several cell types by immunofluorescence and immunoelectron microscopy. Man II was most commonly found in medial- and/or trans- cisternae but showed cell type-dependent variations in intra-Golgi distribution. It was variously localized to either medial (NRK and CHO cells), both medial and trans (pancreatic acinar cells, enterocytes), or trans- (goblet cells) cisternae, or distributed across the entire Golgi stack (hepatocytes and some enterocytes). The distribution of Man I largely coincided with that of Man II in that it was detected primarily in medial- and trans-cisternae. It also showed cell type dependent variations in its intra-Golgi distribution. Man I and Man II were also detected within secretory granules and at the cell surface of some cell types (enterocytes, pancreatic acinar cells, goblet cells). In the case of Man II, cell surface staining was shown not to be due to antibody cross-reactivity with oligosaccharide epitopes. These results indicate that the distribution of Man I and Man II within the Golgi stack of a given cell type overlaps considerably, and their distribution from one cell type to another is more variable and less compartmentalized than previously assumed

Mucin-type O-glycosylation is controlled by short- and long-range glycopeptide substrate recognition that varies among members of the polypeptide GalNAc transferase family

A large family of UDP-GalNAc:polypeptide GalNAc transferases (ppGalNAc-Ts) initiates and defines sites of mucin-type Ser/Thr-O-GalNAc glycosylation. Family members have been classified into peptide- and glycopeptide-preferring subfamilies, although both families possess variable activities against glycopeptide substrates. All but one isoform contains a C-terminal carbohydrate-binding lectin domain whose roles in modulating glycopeptide specificity is just being understood. We have previously shown for several peptide-preferring isoforms that the presence of a remote Thr-O-GalNAc, 6–17 residues from a Ser/Thr acceptor site, may enhance overall catalytic activity in an N- or C-terminal direction. This enhancement varies with isoform and is attributed to Thr-O-GalNAc interactions at the lectin domain. We now report on the glycopeptide substrate utilization of a series of glycopeptide (human-ppGalNAc-T4, T7, T10, T12 and fly PGANT7) and peptide-preferring transferases (T2, T3 and T5) by exploiting a series of random glycopeptide substrates designed to probe the functions of their catalytic and lectin domains. Glycosylation was observed at the −3, −1 and +1 residues relative to a neighboring Thr-O-GalNAc, depending on isoform, which we attribute to specific Thr-O-GalNAc binding at the catalytic domain. Additionally, these glycopeptide-preferring isoforms show remote lectin domain-assisted Thr-O-GalNAc enhancements that vary from modest to none. We conclude that the glycopeptide specificity of the glycopeptide-preferring isoforms predominantly resides in their catalytic domain but may be further modulated by remote lectin domain interactions. These studies further demonstrate that both domains of the ppGalNAc-Ts have specialized and unique functions that work in concert to control and order mucin-type O-glycosylation

Glycoengineering of NK Cells with Glycan Ligands of CD22 and Selectins for B-Cell Lymphoma Therapy

CD22, a member of Siglec family of sialic acid binding proteins, has restricted expression on B cells. Antibody-based agents targeting CD22 or CD20 on B lymphoma and leukemia cells exhibit clinical efficacy for treating these malignancies, but also attack normal B cells leading to immune deficiency. Here, we report a chemoenzymatic glycocalyx editing strategy to introduce high-affinity and specific CD22 ligands onto NK-92MI and cytokine-induced killer cells to achieve tumor-specific CD22 targeting. These CD22-ligand modified cells exhibited significantly enhanced tumor cell binding and killing in vitro without harming healthy B cells. For effective lymphoma cell killing in vivo we further functionalized CD22 ligand-modified NK-92MI cells with the E-selectin ligand sialyl Lewis X to promote trafficking to bone marrow. The dual-functionalized cells resulted in the efficient suppression of B lymphoma in a xenograft model. Our results suggest that nature killer cells modified with glycan ligands to CD22 and selectins promote both targeted killing of B lymphoma cells and improved trafficking to sites where the cancer cells reside, respectively

Short Linear Motifs: Ubiquitous and Functionally Diverse Protein Interaction Modules Directing Cell Regulation

The eukaryotic cell is a bustling collection of macromolecules acting cooperatively to mediate the functions required for cell viability. Specific, context-dependent and tightly controlled physical interactions between these cellular components govern the necessary physiological processes, from cell division to cell death. The specificity, conditionality, and regulation of these binding events depend on communication between the interacting molecules and their surroundings. For proteins, most of this communication is mediated by a variety of modules that are embedded within the protein sequence, can bind a wide array of ligands, and have catalytic, regulatory, or scaffolding activity. These functional units enable proteins to sense, integrate, and transmit environmental and cell state indicators and concomitantly instigate cellular decisions based on the information available to the system