Assignment of O-Glycan Attachment Sites to the Hinge-like Regions of Human Lysosomal Membrane Glycoproteins Lamp-1 and Lamp-2

130

Melanin pigments are synthesized within specialized organelles called melanosomes and polymerize on intraluminal fibrils that form within melanosome precursors. The fibrils consist of proteolytic fragments derived from Pmel17, a pigment cell-specific integral membrane protein. The intracellular pathways by which Pmel17 accesses melanosome precursors and the identity of the Pmel17 derivatives within fibrillar melanosomes have been a matter of debate. We show here that antibodies that detect Pmel17 within fibrillar melanosomes recognize only the luminal products of proprotein convertase cleavage and not the remaining products linked to the transmembrane domain. Moreover, antibodies to the N and C termini detect only Pmel17 isoforms present in early biosynthetic compartments, which constitute a large fraction of detectable steady state Pmel17 in cell lysates because of slow early biosynthetic transport and rapid consumption by fibril formation. Using an antibody to a luminal epitope that is destroyed upon modification by O-linked oligosaccharides, we show that all post-endoplasmic reticulum Pmel17 isoforms are modified by Golgi-associated oligosaccharide transferases, and that only processed forms contribute to melanosome biogenesis. These data indicate that Pmel17 follows a single biosynthetic route from the endoplasmic reticulum through the Golgi complex and endosomes to melanosomes, and that only fragments encompassing previously described functional luminal determinants are present within the fibrils. These data have important implications for the site and mechanism of fibril formation.

Section: Glycosylation and Antibody Reactivity Of Pmel In Cells That supporting

confidence: 54%

Premelanosome Amyloid-like Fibrils Are Composed of Only Golgi-processed Forms of Pmel17 That Have Been Proteolytically Processed in Endosomes

Harper

Theos

Herman

et al. 2008

130

“…Non-␣-helical and non-␤-sheet structure satisfies such a requirement. In addition, it is most likely that such an amino acid sequence does not take a particular conformation, and its structure is flexible (49). It is thus possible that MSD may function as a hinge region where Oglycans are attached.…”

Section: Discussionmentioning

confidence: 99%

“…Since the amino acid sequence of MSD is enriched with proline, threonine, and serine, MSD most likely does not have a unique conformation, such as an ␣-helical or ␤-sheet structure (49). Since O-glycosylation takes place in the Golgi apparatus, O-glycosylation sites need to be exposed to the environment after a protein folds.…”

Section: Discussionmentioning

confidence: 99%

Biosynthesis of HNK-1 Glycans on O-Linked Oligosaccharides Attached to the Neural Cell Adhesion Molecule (NCAM)

Ong¹,

Suzuki

Bélot³

et al. 2002

The HNK-1 glycan, sulfo33GlcA␤133Gal␤134-GlcNAc␤13 R, is highly expressed in neuronal cells and apparently plays critical roles in neuronal cell migration and axonal extension. The HNK-1 glycan synthesis is initiated by the addition of ␤1,3-linked GlcA to Nacetyllactosamine followed by sulfation of the C-3 position of GlcA. The cDNAs encoding ␤1,3-glucuronyltransferase (GlcAT-P) and HNK-1 sulfotransferase (HNK-1ST) have been recently cloned. Among various adhesion molecules, the neural cell adhesion molecule (NCAM) was shown to contain HNK-1 glycan on N-glycans. In the present study, we first demonstrated that NCAM also bears HNK-1 glycan attached to O-glycans when NCAM contains the O-glycan attachment scaffold, musclespecific domain, and is synthesized in the presence of core 2 ␤1,6-N-acetylglucosaminyltransferase, GlcAT-P, and HNK-1ST. Structural analysis of the HNK-1 glycan revealed that the HNK-1 glycan is attached on core 2 branched O-glycans, sulfo33GlcA␤133Gal␤134-GlcNAc␤136(Gal␤133)GalNAc. Using synthetic oligosaccharides as acceptors, we found that GlcAT-P and HNK-1ST almost equally act on oligosaccharides, mimicking N-and O-glycans. By contrast, HNK-1 glycan was much more efficiently added to N-glycans than O-glycans when NCAM was used as an acceptor. These results are consistent with our results showing that HNK-1 glycan is minimally attached to O-glycans of NCAM in fetal brain, heart, and the myoblast cell line, C2C12. These results combined together indicate that HNK-1 glycan can be synthesized on core 2 branched O-glycans but that the HNK-1 glycan is preferentially added on N-glycans over O-glycans of NCAM, probably because N-glycans are extended further than O-glycans attached to NCAM containing the muscle-specific domain.

“…There is little architectural similarity between SIMPLE and classic bipartite or semi-bipartite patterns of the extracellular domain of LAMP or the Endolyn family (41,43). Mucin-like domains and hinge regions of the above families of proteins are heavily Nand O-glycosylated (41,44), whereas SIMPLE is completely devoid of glycosylation. The C-terminal domain also does not show any similarity except for the presence of dileucine (LL) and YXX⌽ motifs, one of which is invariably present in the cytoplasmic domain of the above-mentioned families.…”

Section: Discussionmentioning

confidence: 99%

Mycobacterium bovis Bacillus Calmette-Guerin and Its Cell Wall Complex Induce a Novel Lysosomal Membrane Protein, SIMPLE, That Bridges the Missing Link between Lipopolysaccharide and p53-inducible Gene, LITAF(PIG7), and Estrogen-inducible Gene, EET-1

Moriwaki

Begum

Kobayashi

et al. 2001

121

LITAF and PIG7 encode an identical protein, and they have recently been reported as lipopolysaccharide and p53-inducible genes, respectively. By using the differential display approach, we identified a Mycobacterium bovis BCG cell wall skeleton (BCG-CWS)-inducible gene fragment from human monocytes, showing no homology to any reported gene. Full-length cloning of this fragment reveals the following. 1) The differential display product represents the incomplete 3-untranslated region of LITAF/PIG7. 2) The coding region of the transcript differs from LITAF/PIG7 due to an absence of a single guanine residue, resulting in a potential translational frameshift. 3) The newly coded protein turns out to be 86% identical and 90% similar to an estrogen-inducible rat gene, EET-1. Repeated analysis, expressed sequence tag search, comparison with homologues, and genome sequence analysis confirmed the absence of the single guanine residue. One interesting feature of this protein is that it possesses the RING domain signature and is predicted to be localized in the nucleus. However, detailed analysis together with experimental evidence suggests it is neither a RING family member nor a nuclear protein. Comparison of a total collection of 18 proteins from various species indicates that proteins of this family are small in size and mainly conserved at the C-terminal domain with a unique motif. We characterize this novel protein as an unglycosylated small integral membrane protein of the lysosome/late endosome (SIMPLE) whose expression is elicited in monocytes by live and heat-killed BCG, BCG cell wall complex, lipopolysaccharide, and tumor necrosis factor-␣. To our knowledge this is the first report of pathogen-associated molecular pattern (PAMP)-induced differential expression of a lysosomal membrane protein presumably involved in apoptosis.