Glycosylation is the most versatile and one of the most abundant protein modifications. It has a structural role as well as diverse functional roles in many specific biological functions, including cancer development, viral and bacterial infections, and autoimmunity. The diverse roles of glycosylation in biological processes are rapidly growing areas of research, however, Glycobiology research is limited by the lack of a technology for rapid analysis of glycan composition of glycoproteins. Currently used methods for glycoanalysis are complex, typically requiring high levels of expertise and days to provide answers, and are not readily available to all researcher.We have developed a lectin array-based method, Qproteome™ GlycoArray kits, for rapid analysis of glycosylation profiles of glycoproteins. Glycoanalysis is performed on intact glycoproteins, requiring only 4-6 h for most analysis types. The method, demonstrated in this manuscript by several examples, is based on binding of an intact glycoprotein to the arrayed lectins, resulting in a characteristic fingerprint that is highly sensitive to changes in the protein's glycan composition. The large number of lectins, each with its specific recognition pattern, ensures high sensitivity to changes in the glycosylation pattern. A set of proprietary algorithms automatically interpret the fingerprint signals to provide a comprehensive glycan profile output.

Heparanase is an endoglycosidase which cleaves heparan sulfate and hence participates in degradation and remodeling of the extracellular matrix. Importantly, heparanase activity correlated with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of heparan sulfate cleavage and remodeling of the extracellular matrix barrier. Heparanase has been characterized as a glycoprotein, yet glycan biochemical analysis was not performed to date. Here, we applied the Qproteome ™ GlycoArray kit to perform glycan analysis of heparanase, and compared the kit results with the more commonly used biochemical analyses. We employed fibroblasts isolated from patients with I-cell disease (mucolipidosis II), fibroblasts deficient of low density lipoprotein receptor-related protein and fibroblasts lacking mannose 6-phosphate receptor, to explore the role of mannose 6-phosphate in heparanase uptake. Iodinated heparanase has been utilized to calculate binding affinity. We provide evidence for hierarchy of binding to cellular receptors as a function of heparanase concentration. We report the existence of a high affinity, low abundant (i.e., low density lipoprotein receptor-related protein, mannose 6-phosphate receptor), as well as a low affinity, high abundant (i.e., heparan sulfate proteoglycan) receptors that mediate heparanase binding, and suggest that these receptors cooperate to establish high affinity binding sites for heparanase, thus maintaining extracellular retention of the enzyme tightly regulated.

The yeast chromatin protein Sin1p͞Spt2p has long been studied, but the understanding of its function has remained elusive. The protein has sequence similarity to HMG1, specifically binds crossing DNA structures, and serves as a negative transcriptional regulator of a small family of genes that are activated by the SWI͞SNF chromatin-remodeling complex. Recently, it has been implicated in maintaining the integrity of chromatin during transcription elongation. Here we present experiments whose results indicate that Sin1p͞Spt2 is required for, and is directly involved in, the efficient recruitment of the mRNA cleavage͞polyadenylation complex. This conclusion is based on the following findings: Sin1p͞Spt2 frequently binds specifically downstream of many ORFs but almost always upstream of the first polyadenylation site. It directly interacts with Fir1p, a component of the cleavage͞polyadenylation complex. Disruption of Sin1p͞Spt2p results in foreshortened poly(A) tracts on mRNA. It is synthetically lethal with Cdc73p, which is involved in the recruitment of the complex. This report shows that a chromatin component is involved in 3 end processing of RNA. S in1p is a yeast chromatin nonhistone protein that has beenshown to function as a negative transcriptional regulator of several genes, including Suc2 (1), Ino1 (2), and SSA3 (3). Activity of the HO promoter, as measured from an HO promoter driving a lacZ gene (4), is also affected by Sin1. Sin1 (also known as spt2 in this context) mutants were identified as suppressors of Ty and insertions in the 5Ј noncoding region of the HIS4 gene (5). Because the negative regulation of these genes is overcome by the SW1͞SNF chromatin-remodeling complex (4, 6, 7) and the C-terminal domain of Sin1p interacts with Swi1p (8), it was suggested that a function of SIN1p is to somehow maintain chromatin compaction at specific loci in the chromatin. Peterson et al. (2) found a functional relationship between the C-terminal domain (CTD) of RNA polymerase II and Sin1p, but these data were not pursued further. Sequence analysis of Sin1p showed sequence similarity in two domains to HMG1 (6, 7), a known chromatin protein. Work from our laboratory showed that Sin1p can bind four-way junction and crossing DNA structures (9), supporting the idea that Sin1p binds DNA as it enters and exits the nucleosome.In the context of a global mapping project, Tong et al. (10) reported that there is a synthetic lethal interaction between sin1 and cdc73, a member of the PAF complex. The PAF complex, which accompanies RNA polymerase II during elongation, was shown to have an important function in 3Ј end formation and in polyadenylation (11-13). In addition, a functional interaction was demonstrated between Sin1p and Hpr1p, which is associated with the PAF complex (14).Most recently, evidence has been presented indicating that Sin1p͞Spt2p plays roles in transcription elongation, chromatin structure and genome stability (15). In that study, synthetic lethal interactions were reported between sin1 and paf1, and betwe...

A protein or protein complex has previously been identified in Saccharomyces cerevisiae which both binds a short DNA sequence in URS1 of HO and interacts with SIN1. SIN1, which has some sequence similarity to mammalian HMG1, is an abundant chromatin protein in yeast and is thought to participate in the transcriptional repression of a specific family of genes. SIN1 binds DNA weakly, though it has no DNA binding specificity. Here we address the nature of the interaction between SIN1 and the specific DNA binding protein(s) to HO DNA. We show that the isolated C-terminal region of SIN1 can interact in vitro with the DNA binding protein, causing a supershift in a gel mobility shift assay. Interestingly, inclusion of the region in SIN1 which contains two acidic sequences, precludes the binding of recombinant protein to the DNA/protein complex.

The yeast SINI protein is a nuclear protein that together with other proteins behaves as a transcriptional repressor of a family of genes. In addition, sinl mutants are defective in proper mitotic chromosome segregation. In an effort to understand the basis for these phenotypes, we employed the yeast two-hybrid system to identify proteins that interact with SINI in vivo. Here we demonstrate that CDC23, a protein known to be involved in sister chromatid separation during mitosis, is able to directly interact with SINM. Furthermore, using recombinant molecules in vitro, we show that the N terminal of SINI is sufficient to bind a portion of CDC23 consisting solely of tetratrico peptide repeats. Earlier experiments identified the C-terminal domain of SINM to be responsible for interaction with a protein that binds the regulatory region of HO, a gene whose transcription is repressed by SINM. Taken together with the results presented here, we suggest that SINM is a chromatin protein having at least a dual function: The N terminal of SINM interacts with the tetratrico peptide repeat domains of CDC23, a protein involved in chromosome segregation, whereas the C terminal of SINM binds proteins involved in transcriptional regulation.