Proteins on the cell surface are frequently glycosylated, and they are essential for cells. Surface glycoproteins regulate nearly every extracellular event, but compared with global analysis of proteins, comprehensive and site-specific analysis of surface glycoproteins is much more challenging and dramatically understudied. Here, combining metabolic labeling, click-chemistry and enzymatic reactions, and mass spectrometry-based proteomics, we globally characterized surface glycoproteins from eight popular types of human cells. This integrative and effective method allowed for the identification of 2172 N-glycosylation sites and 1047 surface glycoproteins. The distribution and occurrence of N-glycosylation sites were systematically investigated, and protein secondary structures were found to have a dramatic influence on glycosylation sites. As expected, most sites are located on disordered regions. For the sites with the motif N-!P-C, about one-third of them are located on helix structures, while those with the motif N-!P-S/T prefer strand structures. There is almost no correlation between the number of glycosylation sites and protein length, but the number of sites corresponds well with the frequencies of the motif. Quantification results reveal that besides cell-specific glycoproteins, the uniqueness of each cell type further arises from differential expression of surface glycoproteins. The current research indicates that multiple surface glycoproteins including their abundances need to be considered for cell classification rather than a single cluster of differentiation (CD) protein normally used in conventional methods. These results provide valuable information to the *

Small heat-shock proteins (sHSPs) are a widely expressed family of ATP-independent molecular chaperones that are among the first responders to cellular stress. Mechanisms by which sHSPs delay aggregation of client proteins remain undefined. sHSPs have high intrinsic disorder content of up to ~60% and assemble into large, polydisperse homo- and hetero-oligomers, making them challenging structural and biochemical targets. Two sHSPs, HSPB4 and HSPB5, are present at millimolar concentrations in eye lens, where they are responsible for maintaining lens transparency over the lifetime of an organism. Together, HSPB4 and HSPB5 compose the hetero-oligomeric chaperone known as α-crystallin. To identify the determinants of sHSP function, we compared the effectiveness of HSPB4 and HSPB5 homo-oligomers and HSPB4/HSPB5 hetero-oligomers in delaying the aggregation of the lens protein γD-crystallin. In chimeric versions of HSPB4 and HSPB5, chaperone activity tracked with the identity of the 60-residue disordered N-terminal regions (NTR). A short 10-residue stretch in the middle of the NTR (“Critical sequence”) contains three residues that are responsible for high HSPB5 chaperone activity toward γD-crystallin. These residues affect structure and dynamics throughout the NTR. Abundant interactions involving the NTR Critical sequence reveal it to be a hub for a network of interactions within oligomers. We propose a model whereby the NTR critical sequence influences local structure and NTR dynamics that modulate accessibility of the NTR, which in turn modulates chaperone activity.

Small heat shock proteins (sHSPs) are chaperones whose importance in protein homeostasis is exemplified by dozens of missense mutations associated with tissue-specific disease states. Despite decades of studies, the structure, dynamics, and mechanism of chaperone activity remain unclear. Here we show that the human sHSP HSPB5 distinguishes native lens protein γD-crystallin from damaged γD-crystallin even though the mutant/damaged client is folded. The disordered N-terminal region of HSPB5 (NTR) is essential for its chaperone activity, whereas the structured domain (ACD) has no intrinsic activity. Nevertheless, two sHSP mutational hotspots associated with disease, D109 and R120, are located in the ACD. Our studies on wild-type HSPB5 oligomers reveal that distinct regions within the NTR interact with specific grooves presented on the ACD dimer and/or with other NTR sub-regions and that the number of binding partners is greater than the number of binding sites, leading to a large, but finite number of potential combinations of interactions at any given time. The ACD mutations result in increased dynamics and accessibility of the disordered NTR and enhanced chaperone activity in vitro. Our findings reveal that HSPB5 quasi-order is delicately balanced and that perturbations arising from mutations within the structured core cause alterations that contribute to misbalance in eye lens protein homeostasis that lead to cataract formation.

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