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The hyaluronic acid receptor for endocytosis (HARE)/Stabilin-2 is the primary systemic scavenger receptor for 13 ligands including hyaluronan (HA), heparin and chondroitin sulfates. Most ligand-binding sites are within the 190 kDa isoform, which contains ∼25 kDa of N-glycans and is the C-terminal half of the full-length 315 kDa HARE. Glycoproteomic analyses of purified recombinant human 190-HARE ecto-domain identified a diverse population of glycans at 10 of 17 consensus sites. The most diversity (and the only sialylated structures) occurred at N2280, within the HA-binding Link domain. To determine if these N-glycans are required for HA binding, we created human Flp-In 293 cell lines expressing membrane-bound or soluble ecto-domain variants of 190-HARE(N2280A). Membrane-bound HARE lacking Link domain N-glycans mediated rapid HA endocytosis, but purified 190-HARE(N2280A) ecto-domain showed little or no HA binding in ELISA-like, HA-HARE pull-down assays or by surface plasmon resonance analysis (which detected very high apparent affinity for 190-HARE ecto-domain binding to HA; Kd = 5.2 nM). The results indicate that Link domain N-glycans stabilize interactions that facilitate HA binding to HARE. [ABSTRACT FROM PUBLISHER]

CD52 is composed of a 12 amino acid peptide with N-linked glycans bound to the single potential glycosylation site at position 3, and a glycosylphosphatidylinositol-anchor attached at the C-terminus. Some glycoforms of this molecule expressed in the male reproductive tract are recognized by complement-dependent sperm-immobilizing antibodies in infertile patients making this antigen an important target for immunocontraception and fertility studies. Although the amount of posttranslational modification is already remarkable for such a small polypeptide, O-glycosylation of CD52 has additionally been implicated by several studies, but never rigorously characterized. In this report, we show clear evidence for the presence of O-glycans in CD52 preparations immunopurified using the murine S19 monoclonal antibody generated against sperm agglutination antigen-1 (SAGA-1), a male reproductive tract specific form of CD52. The O-glycans have been characterized by MALDI-TOF and tandem mass spectrometry after reductive elimination and permethylation. The data indicate that the major SAGA-1 O-glycans are core 1 and 2 mucin-type structures, with and without sialic acid (NeuAc0−2Hex1−3HexNAc1−2HexNAcitol). Minor fucosy- lated O-glycans are also present including some struc- tures with putative Ley epitopes (NeuAc0−1Fuc1−3Hex1−2 HexNAc0−1HexNAcitol). Analysis of O-glycopeptides by tandem mass spectrometry provided an additional level of support for the O-glycosylation of SAGA-1. Elucidation of the O-glycosylation of SAGA-1 adds to the complexity of this molecule and may help to explain its biological activity. [ABSTRACT FROM PUBLISHER]

The biochemical and biophysical properties of mucinsare largely determined by extensive O-glycosylation of serine- andthreonine-rich tandem repeat (TR) domains. In a number of humandiseases aberrant O-glycosylation is associated with variationsin the properties of the cell surface–associated and secretedmucins. To evaluate in vivo the O-glycosylationof mucin TR domains, we generated recombinant chimeric mucins withTR sequences from MUC2, MUC4, MUC5AC, or MUC5B, which were substituted forthe native TRs of epitope-tagged MUC1 protein (MUC1F). These hybridmucins were extensively O-glycosylated and showed the expected associationwith the cell surface and release into culture media. The presenceof different TR domains within the chimeric mucins appears to have limitedinfluence on their posttranslational processing. Alterations inglycosylation were detailed by fast atom bombardment mass spectrometryand reactivity with antibodies against particular blood-group andtumor-associated carbohydrate antigens. Future applications of thesechimeras will include investigations of mucin posttranslationalmodification in the context of disease. [ABSTRACT FROM PUBLISHER]