Hyaluronan-binding Motifs Research Articles

Abstract 1857: Development of monoclonal antibodies against CD44v specifically expressed in cancers

Abstract CD44 is a transmembrane glycoprotein that binds to the extracellular matrix including hyaluronic acid. Human CD44 has 19 exons, 10 of which are commonly present in all isoforms. CD44 standard isoform (CD44s) is composed of the common exons, and is expressed in many cell types. CD44 variants (CD44v) are produced by alternative splicing, in which the remaining nine variant exons are inserted into the CD44 standard isoform in various combinations. The CD44 isoforms have overlapping and unique functions. Both CD44s and CD44v harbor hyaluronan-binding motifs that promote interaction with the tumor microenvironment, which activate various signaling pathways in tumor cells. CD44v is mainly expressed in epithelial cells and is involved in cell adhesion, proliferation, and survival. Overexpression of CD44v is observed in many solid tumors and plays an important role in invasion, metastasis, and acquisition of stemness. In this study, we employed the Cell-Based Immunization and Screening method (CBIS method), and established highly sensitive anti-CD44 monoclonal antibodies (mAbs) using CD44v3-10-overexpressed cell lines as immunogens. After that, we determined the epitopes by enzyme-linked immunosorbent assay and investigated their applications. As a result, we successfully established eight mAbs against each CD44v (v3 to v10), which can be used for flow cytometry, western blotting, and immunohistochemistry. Furthermore, some of them exhibited cancer specificity against pancreatic cancers, colorectal cancers, gastric cancers, or oral cancers, indicating that established anti-CD44v mAbs are useful for the antibody therapies. Citation Format: Yukinari Kato, Hiroyuki Suzuki, Mika K. Kaneko. Development of monoclonal antibodies against CD44v specifically expressed in cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1857.

Hyaluronan-binding peptide for targeting peritoneal carcinomatosis.

Peritoneal carcinomatosis results from dissemination of solid tumors in the peritoneal cavity, and is a common site of metastasis in patients with carcinomas of gastrointestinal or gynecological origin. Peritoneal carcinomatosis treatment is challenging as poorly vascularized, disseminated peritoneal micro-tumors are shielded from systemic anticancer drugs and drive tumor regrowth. Here, we describe the identification and validation of a tumor homing peptide CKRDLSRRC (IP3), which upon intraperitoneal administration delivers payloads to peritoneal metastases. IP3 peptide was identified by in vivo phage display on a mouse model of peritoneal carcinomatosis of gastric origin (MKN-45P), using high-throughput sequencing of the peptide-encoding region of phage genome as a readout. The IP3 peptide contains a hyaluronan-binding motif, and fluorescein-labeled IP3 peptide bound to immobilized hyaluronan in vitro. After intraperitoneal administration in mice bearing peritoneal metastases of gastric and colon origin, IP3 peptide homed robustly to macrophage-rich regions in peritoneal tumors, including poorly vascularized micro-tumors. Finally, we show that IP3 functionalization conferred silver nanoparticles the ability to home to peritoneal tumors of gastric and colonic origin, suggesting that it could facilitate targeted delivery of nanoscale payloads to peritoneal tumors. Collectively, our study suggests that the IP3 peptide has potential applications for targeting drugs, nanoparticles, and imaging agents to peritoneal tumors.

Hyaluronan Binding Motifs of USP17 and SDS3 Exhibit Anti-Tumor Activity

BackgroundWe previously reported that the USP17 deubiquitinating enzyme having hyaluronan binding motifs (HABMs) interacts with human SDS3 (suppressor of defective silencing 3) and specifically deubiquitinates Lys-63 branched polyubiquitination of SDS3 resulting in negative regulation of histone deacetylase (HDAC) activity in cancer cells. Furthermore, USP17 and SDS3 mutually interact with each other to block cell proliferation in HeLa cells but the mechanism for this inhibition in cell proliferation is not known. We wished to investigate whether the HABMs of USP17 were responsible for tumor suppression activity.Methodology/Principal FindingsSimilarly to USP17, we have identified that SDS3 also has three consecutive HABMs and shows direct binding with hyaluronan (HA) using cetylpyridinium chloride (CPC) assay. Additionally, HA oligosaccharides (6-18 sugar units) competitively block binding of endogenous HA polymer to HA binding proteins. Thus, administration of HA oligosaccharides antagonizes the interaction between HA and USP17 or SDS3. Interestingly, HABMs deleted USP17 showed lesser interaction with SDS3 but retain its deubiquitinating activity towards SDS3. The deletion of HABMs of USP17 could not alter its functional regulation on SDS3-associated HDAC activity. Furthermore, to explore whether HABMs in USP17 and SDS3 are responsible for the inhibition of cell proliferation, we investigated the effect of USP17 and SDS3-lacking HABMs on cell proliferation by soft agar, apoptosis, cell migration and cell proliferation assays.ConclusionsOur results have demonstrated that these HABMs in USP17 and its substrate SDS3 are mainly involved in the inhibition of anchorage-independent tumor growth.

Polyclonal and Monoclonal Antibodies Specific for USP17, a Proapoptotic Deubiquitinating Enzyme

USP17, a deubiquitinating enzyme (DUB), belongs to the ubiquitin-specific protease protein family. USP17 has highly conserved Cys, His, and Asp domains responsible for the deubiquitinating activity, and two hyaluronan binding motifs in its sequence. We reported that the presence of hyaluronan binding motifs in USP17 is responsible for the cell viability of cancerous cells. Although many if not all of the DUBs contain hyaluronan binding motifs in its sequence, the actual mechanism of hyaluronan binding motifs regulating signal transduction remains unclear. USP17 also regulates Ras activation and cell proliferation by inhibiting RCE1 activity. Thus USP17 may have a critical role in regulating tumor pathway through its deubiquitinating activity on various tumor-related proteins. This study reports the generation and characterization of polyclonal and six monoclonal antibodies against USP17 through immunoblotting, immunoprecipitation, and immunofluorescence microscopy analyses. These antibodies of USP17 will be useful as tools to investigate the mechanism of hyaluronan-mediated tumor suppression and also to screen interacting oncogenic substrates by immunoprecipitation assay.

Competitive binding of heparin with hyaluronan to a specific motif in SPACR

The critical hyaluronan binding motif (HABM) in sialoprotein associated with cones and rods (SPACR) has already been determined. As sialoproteoglycan associated with cones and rods, another interphotoreceptor matrix molecule, binds to chondroitin sulfate and heparin with or without the employment of HABMs, respectively, we evaluated and compared the binding of these glycosaminoglycans to SPACR. A western blotting study in combination with inhibition assays showed that heparin bound specifically to SPACR. A series of GST fusion proteins covering the whole SPACR molecule narrowed down the region responsible for the binding. Finally, a site-directed mutagenesis assay demonstrated that the critical HABM also acts as a specific binding site for heparin. These results were supported with mutual inhibitions by hyaluronan and heparin in analyses using GST fusion proteins and native SPACR derived from retina. Thus, these glycosaminoglycans bind to SPACR in a different manner than to sialoproteoglycan associated with cones and rods. The competitive binding between hyaluronan and heparin to SPACR, mediated through the identical HABM, may dominate the functions of SPACR, in turn involving physiological and pathological processes involved in retinal development, aging and other related disorders.

Hyaluronan- and RNA-binding deubiquitinating enzymes of USP17 family members associated with cell viability

BackgroundProtein degradation by the ubiquitin system plays a crucial role in numerous cellular signaling pathways. Deubiquitination, a reversal of ubiquitination, has been recognized as an important regulatory step in the ubiquitin-dependent degradation pathway.ResultsWhile identifying putative ubiquitin specific protease (USP) enzymes that contain a conserved Asp (I) domain in humans, 4 USP17 subfamily members, highly homologous to DUB-3, have been found (USP17K, USP17L, USP17M, and USP17N), from human chorionic villi. Expression analysis showed that USP17 transcripts are highly expressed in the heart, liver, and pancreas and are expressed moderately in various human cancerous cell lines. Amino acid sequence analysis revealed that they contain the highly conserved Cys, His, and Asp domains which are responsible for the deubiquitinating activity. Biochemical enzyme assays indicated that they have deubiquitinating activity. Interestingly, the sequence analysis showed that these proteins, with exception of USP17N, contain the putative hyaluronan/RNA binding motifs, and cetylpyridinium chloride (CPC)-precipitation analysis confirmed the association between these proteins and intracellular hyaluronan and RNA.ConclusionHere, we report that the overexpression of these proteins, with exception of USP17N, leads to apoptosis, suggesting that the hyaluronan and RNA binding motifs in these enzymes play an important role in regulating signal transduction involved in cell death.

Hyaluronan-binding motif identified by panning a random peptide display library

The glycosaminoglycan hyaluronan (HA) is involved in a variety of functions such as cell migration, adhesion, activation of intracellular signaling, metastasis, inflammation and wound repair. These functions of HA are mediated via HA-binding proteins (HABPs). To derive details of the HA-binding site in HABPs, here, we panned a random peptide display library expressed on the E. coli flagellin protein using HA-coated plates. Using this random peptide display library, 40 positive clones were obtained and the nucleotide sequences were determined. As a result, an Arg-Arg sequence, in addition to the known B-X7-B motif, was found to bind to HA. A binding experiment using the IAsys resonant mirror biosensor verified that a peptide containing an Arg-Arg sequence binds to HA.

Aberrant Intronic Splicing of the Hyaluronan Synthase 1 Gene (HAS1) in Multiple Myeloma: A Biologically Relevant Marker That Predicts for Poor Survival.

Hyaluronan synthases, plasma membrane proteins encoded by the HAS genes, synthesize different sizes of hyaluronan (HA), an extracellular matrix molecule which is biologically active in malignant spread and signaling. HA is also a ligand for RHAMM, a receptor, shown by our laboratory to regulate mitotic events. We demonstrated that abnormal expression of RHAMM in model systems results in mitotic abnormalities that may lead to chromosomal missegregation in multiple myeloma (MM). In MM patients we detected overexpression of HAS1 transcripts and we have identified three aberrantly spliced variants of HAS1 designated as HAS1Va, Vb, and Vc. The statistical analysis of samples from 58 MM patients taken at the time of diagnosis showed that expression of HAS1Vb either alone or in combination with HAS1 and variants in MM cells strongly correlates with poor survival (P=0.001). This expression analysis of HAS1 and variants in MM patients suggests that, particularly HAS1Vb may contribute to early myelomagenesis since these transcripts are detected individually or in combination with other HAS1 variants in MM and MGUS patients at the time of diagnosis. Longitudinal analysis of HAS1 and its variants in 18 unselected MM patients showed expression of the family of HAS1 transcripts in a majority of these patients at diagnosis (65% of patients) and relapse (71.4% of patients). All three HAS1 variants are truncated as the result of exon skipping and/or intron retention. In general, partial retention of introns appears to be characteristic of the genes associated with a malignant phenotype. Alignment and protein motif screening analysis showed that all three variants of HAS1 retain the motif, which is responsible for the synthesis of HA. In silico analysis demonstrated that HAS1 variants are able to fold appropriately. Protein expression of HAS1 variants was detected by western blotting using lysates from MM cell lines. Enzymatic activity of family of HAS1 proteins was verified by a particle exclusion assay (PEA) and HA staining. Using these methods we detect HA matrix and intracellular HA around and in MM cells. Based on PEA analysis, both HAS1Va and HAS1Vb appear to synthesize extracellular HA. Synthesis of extracellular HA by MM cells may impact disease biology by contributing to drug resistance and could accommodate spread of MM cells by facilitating motility and malignant spread. Furthermore, HAS1Vb, the only variant with strong clinical impact, also appears to be the only splice variant that produces intracellular HA, a form of HA that may modulate RHAMM associations with the mitotic spindle. The HA binding motif of RHAMM overlaps with its centrosomal targeting domain. Strong perinuclear localization of intracellular HA, which branches out from the perinuclear compartment toward the MM cell plasma membrane suggests that these molecules may also contribute to the maintenance of cellular architecture by malignant MM cells. We speculate that at least part of the strong clinical impact of aberrant HAS1Vb intronic splicing reflects the predicted ability of intracellular HA synthesized by HAS1Vb to modulate the function of RHAMM by inhibiting centrosomal targeting, thereby promoting aberrant mitosis. If this working hypothesis is correct, HAS1 and its variants, particularly HAS1Vb, in concert with RHAMM may be key contributors to chromosomal instability in MM. Funded by CIHR and by CA80963 from NCI.

Cloning and molecular characterization of the first aquatic hyaluronidase, SFHYA1, from the venom of stonefish ( Synanceja horrida)

We report here for the first time the molecular characterization of a hyaluronidase from an aquatic source. SFHYA1 is the hyaluronidase found in the venom gland of stonefish, Synanceja horrida. Using a cDNA segment amplified with degenerate oligonucleotides based on the amino acid sequences of a conserved region in testicular-type hyaluronidases and a tryptic fragment of SFHYA1, clones encoding the precursor of this enzyme were isolated from a cDNA library prepared from stonefish venom glands. The deduced amino acid sequence of SFHYA1 shows that SFHYA1 is expressed as a precursor peptide with a 28-residue signal peptide for targeting it into endoplasmic reticulum. Mature SFHYA1 is a polypeptide composed of 449 residues containing three potential N-glycosylation sites, four putative hyaluronan-binding motifs [B(X) 7B] and various residues implicated in substrate binding and catalysis. This cDNA was expressed in an active form in insect-cells but not in E. coli. Homology-based computational analyses suggested that SFHYA1 closely resembles the PH-20 family of hyaluronidases.

Spacrcan Binding to Hyaluronan and Other Glycosaminoglycans: MOLECULAR AND BIOCHEMICAL STUDIES

Photoreceptors project from the outer retinal surface into a specialized glycocalyx, the interphotoreceptor matrix (IPM), which contains hyaluronan (HA) and two novel proteoglycans, Spacr and Spacrcan. This matrix must be stable enough to function in the attachment of the retina to the outer eye wall yet porous enough to allow movement of metabolites between these tissues. How this matrix is organized is not known. HA is a potential candidate in IPM organization since biochemical studies show that these proteoglycans bind HA. RHAMM (receptor for HA-mediated motility)-type HA binding motifs (HABMs) are present in their deduced amino acid sequence and may be the sites of this HA interaction. To test this hypothesis, we subcloned three fragments of mouse Spacrcan that contain the putative HABMs. We found that each recombinant fragment binds HA. Binding decreased when residues in the HABMs were mutated. This provides direct evidence that the RHAMM-type HABMs in Spacrcan are involved in hyaluronan binding. Since chondroitin sulfate and heparan sulfate proteoglycans are important for retinal development and function, we also evaluated the binding of these recombinant proteins to heparin and chondroitin sulfates, the glycosaminoglycan side chain of these proteoglycans. We found that each recombinant protein bound to both heparin and chondroitin sulfates. Binding to chondroitin sulfates involved these HABMs, because mutagenesis reduced binding. Binding to heparin was probably not mediated through these HABMs since heparin binding persisted following their mutagenesis. These studies provide the first evidence defining the sites of protein-carbohydrate interaction of molecules present in the IPM.

SPACRCAN in the interphotoreceptor matrix of the mouse retina: molecular, developmental and promoter analysis

SPACRCAN 1 1 Human SPACRCAN gene is designated by uppercase italicized letters. Mouse SPACRCAN gene is designated by italicized letters with uppercase for the initial letter and lowercase for the rest. SPACRCAN protein is designated by uppercase letters in standard fonts. is a novel proteoglycan present in the interphotoreceptor matrix (IPM) of the rat and human retina that resists aqueous extraction through its binding to hyaluronan. The purpose of this study was: to clone mouse Spacrcan; to characterize the promoter elements; to define the deduced amino acid sequence; to establish the time of Spacrcan expression during retinal development; and to determine the time of appearance and distribution of SPACRCAN protein. Spacrcan cDNA clone was obtained through PCR amplification of a mouse retina cDNA library, and RT-PCR amplification and 5′RACE of mouse retina RNA. The deduced polypeptide sequence of mouse SPACRCAN contains a signal peptide at the N-terminal, seven N-link glycosylation sites, numerous potential O-linked glycosylation sites in a central mucin-like domain, two glycosaminoglycan attachment sites, five potential hyaluronan-binding motifs, two epidermal growth factor-like domains, and a hydrophobic stretch of 23 amino acids near the C-terminal. Comparison of the genomic structure of mouse and human SPACRCAN showed significant structure conservation. Analysis of the promoter region revealed several important putative regulatory elements including a Ret-1/PCE-1 element, an 11 base motif for Crx binding, six copies of PIRE, a Ret-4 element, three copies of AP-1, a CRE element, and five copies of GATA3. Northern blot analysis and immunohistochemistry were used to determine the tissue specificity of Spacrcan mRNA and to localize SPACRCAN in developing retina. Spacrcan mRNA is expressed in both retina and pineal gland and was detectable as early as embryonic day 15. The protein is first detectable in the IPM at postnatal day 8 where it increases in concert with the extension of photoreceptor inner and outer segments from the outer retinal surface. The presence of several unique regulatory elements in the promoter region and characteristic molecular features shared with the orthologue in human and rat suggest an important functional role of SPACRCAN in the IPM. The time of appearance of the SPACRCAN protein during retinal development suggests that this matrix protein may establish the extracellular microenvironment into which photoreceptor outer segments are elaborated.

Stabilin-1 and −2 constitute a novel family of fasciclin-like hyaluronan receptor homologues

MS-1, a high-molecular-mass protein expressed by non-continuous and angiogenic endothelial cells and by alternatively activated macrophages (Mφ2), and the hepatic sinusoidal endothelial hyaluronan clearance receptor are similar with respect to tissue distribution and biochemical characteristics. In the present study we purified these proteins by immuno- and hyaluronan-affinity chromatography respectively, sequenced tryptic peptides and generated full-length cDNA sequences in both mouse and human. The novel genes, i.e. stabilin-1 and stabilin-2, code for homologous transmembrane proteins featuring seven fasciclin-like adhesion domains, 18–20 epidermal-growth-factor domains, one X-link domain and three to six B-(X7)-B hyaluronan-binding motifs. Northern-blotting experiments revealed the presence of both stabilins in organs with predominant endothelial sinuses such as liver, spleen and lymph node: stabilin-1 mRNA was also detected in organs with predominant Mφ2 cells, such as placenta, and in interleukin-4/glucocorticoid-stimulated Mφ2 cells in vitro. A polyclonal antibody made against human recombinant stabilin-1 confirmed the expression of stabilin-1 protein in splenic sinus endothelial cells in vivo and in Mφ2 in vitro. On the basis of high similarity at the protein level and the unique domain composition, which differs from that of all other known fasciclin-like proteins and hyaluronan receptors, stabilin-1 and stabilin-2 define a novel family of fasciclin-like hyaluronan receptor homologues that might play a role in cell—cell and cell—matrix interactions in vascular function and inflammatory processes.

Stabilin-1 and -2 constitute a novel family of fasciclin-like hyaluronan receptor homologues.

MS-1, a high-molecular-mass protein expressed by non-continuous and angiogenic endothelial cells and by alternatively activated macrophages (Mphi2), and the hepatic sinusoidal endothelial hyaluronan clearance receptor are similar with respect to tissue distribution and biochemical characteristics. In the present study we purified these proteins by immuno- and hyaluronan-affinity chromatography respectively, sequenced tryptic peptides and generated full-length cDNA sequences in both mouse and human. The novel genes, i.e. stabilin-1 and stabilin-2, code for homologous transmembrane proteins featuring seven fasciclin-like adhesion domains, 18-20 epidermal-growth-factor domains, one X-link domain and three to six B-(X(7))-B hyaluronan-binding motifs. Northern-blotting experiments revealed the presence of both stabilins in organs with predominant endothelial sinuses such as liver, spleen and lymph node: stabilin-1 mRNA was also detected in organs with predominant Mphi2 cells, such as placenta, and in interleukin-4/glucocorticoid-stimulated Mphi2 cells in vitro. A polyclonal antibody made against human recombinant stabilin-1 confirmed the expression of stabilin-1 protein in splenic sinus endothelial cells in vivo and in Mphi2 in vitro. On the basis of high similarity at the protein level and the unique domain composition, which differs from that of all other known fasciclin-like proteins and hyaluronan receptors, stabilin-1 and stabilin-2 define a novel family of fasciclin-like hyaluronan receptor homologues that might play a role in cell-cell and cell-matrix interactions in vascular function and inflammatory processes.

SPACR in the Interphotoreceptor Matrix of the Mouse Retina: Molecular, Biochemical and Immunohistochemical Characterization

Mouse SPACR cDNA was cloned by screening a mouse retina cDNA library using a PCR probe derived from human SPACR cDNA. Mouse SPACR cDNA comprises 3675bp containing an open reading frame coding for 742 amino acids. Multitissue Northern blot analysis and in situ hybridization studies indicate that SPACR expression is restricted to retinal photoreceptors. The SPACR core protein was identified with Western blotting following SDS–PAGE with a SPACR C-terminal peptide polyclonal antibody and a chondroitin-6-sulfate Δdisaccharide monoclonal antibody. The 150kD immunopositive band was isolated, digested with trypsin and the peptides analysed by MALDI mass spectroscopy. Peptide mass mapping confirmed the identity of the 150kD immunopositive band to be mouse SPACR core protein. Alignment comparisons of the deduced amino acid sequence of mouse and human SPACR show 64% homology. Like SPACR in the human interphotoreceptor matrix, the mouse orthologue contains a large central mucin-like domain flanked by consensus sites for N-linked oligosaccharide attachment, one EGF-like domain and four hyaluronan-binding motifs. Unlike human SPACR, which contains no conventional consensus sites for glycosaminoglycan attachment, mouse SPACR contains three. Recent biochemical studies of human and mouse SPACR protein indicate that this novel interphotoreceptor matrix molecule is a glycoprotein in human and a proteoglycan in the mouse. The presence of consensus sites for glycosaminoglycan attachment in the deduced sequence of mouse SPACR and the absence of these sites in human SPACR provide molecular verification of our biochemical results, suggesting that differences in post-translational modifications of SPACR may be important in SPACR function in foveate and non-foveate retinas.

Molecular Characterization of a Novel Intracellular Hyaluronan-binding Protein

Hyaluronan has well defined functions in extracellular matrices and at the surface of cells. However, several studies have now shown that significant pools of hyaluronan are also present intracellularly, but its function therein is unknown. One avenue of investigation that may assist in defining the function of intracellular hyaluronan is to identify intracellular hyaluronan-binding proteins. In previous studies we identified CDC37, a cell cycle regulatory protein, using a monoclonal antibody that recognizes a novel group of hyaluronan-binding proteins. In this study, we have identified a second hyaluronan-binding protein with this antibody and characterized its properties. This protein, which we have termed IHABP4, was also found to be an intracellular and a specific hyaluronan-binding protein, containing several hyaluronan-binding motifs: (R/K)[X(7)](R/K) (where R/K denotes arginine or lysine and X denotes non-acidic amino acids). Furthermore, we have determined the gene organization of IHABP4 and cloned cDNAs for the chick, mouse, and human homologs. Comparison of the deduced chick, mouse, and human protein sequences showed that the hyaluronan-binding motifs, (R/K)[X(7)](R/K), in these sequences are conserved; both chick and mouse IHABP4 were shown directly to bind hyaluronan. Biochemical fractionation and immunofluorescent localization of epitope-tagged IHABP4 indicated that it is mainly present in the cytoplasm. These data support the possibility that intracellular hyaluronan and its binding proteins may play important roles in cell behavior.

SPACRCAN, a Novel Human Interphotoreceptor Matrix Hyaluronan-binding Proteoglycan Synthesized by Photoreceptors and Pinealocytes

The interphotoreceptor matrix is a unique extracellular complex occupying the interface between photoreceptors and the retinal pigment epithelium in the fundus of the eye. Because of the putative supportive role in photoreceptor maintenance, it is likely that constituent molecules play key roles in photoreceptor function and may be targets for inherited retinal disease. In this study we identify and characterize SPACRCAN, a novel chondroitin proteoglycan in this matrix. SPACRCAN was cloned from a human retinal cDNA library and the gene localized to chromosome 3q11.2. Analysis of SPACRCAN mRNA and protein revealed that SPACRCAN is expressed exclusively by photoreceptors and pinealocytes. SPACRCAN synthesized by photoreceptors is localized to the interphotoreceptor matrix where it surrounds both rods and cones. The functional protein contains 1160 amino acids with a large central mucin domain, three consensus sites for glycosaminoglycan attachment, two epidermal growth factor-like repeats, a putative hyaluronan-binding motif, and a potential transmembrane domain near the C-terminal. Lectin and Western blotting indicate an M(r) around 400,000 before and 230,000 after chondroitinase ABC digestion. Removal of N- and O-linked oligosaccharides reduces the M(r) to approximately 160,000, suggesting that approximately 60% of the mass of SPACRCAN is carbohydrate. Finally, we demonstrate that SPACRCAN binds hyaluronan and propose that associations between SPACRCAN and hyaluronan may be involved in organization of the insoluble interphotoreceptor matrix, particularly as SPACRCAN is the major proteoglycan present in this matrix.

SPACR, a Novel Interphotoreceptor Matrix Glycoprotein in Human Retina That Interacts with Hyaluronan

SPACR (sialoprotein associated with cones and rods), is the major 147-150-kDa glycoprotein present in the insoluble interphotoreceptor matrix of the human retina. Immunocytochemistry localizes SPACR to the matrix surrounding rods and cones (Acharya, S., Rayborn, M. E., and Hollyfield, J. G. (1998) Glycobiology 8, 997-1006). From affinity-purified SPACR, we obtained seven peptide sequences showing 100% identity to the deduced sequence of IMPG1, a purported chondroitin 6-sulfate proteoglycan core protein, which binds peanut agglutinin and is localized to the interphotoreceptor matrix. We show here that SPACR is the most prominent 147-150-kDa band present in the interphotoreceptor matrix and is the gene product of IMPG1. SPACR is not a chondroitin sulfate proteoglycan, since it is not a product of chondroitinase ABC digestion and does not react to a specific antibody for chondroitin 6-sulfate proteoglycan. Moreover, the deduced amino acid sequence reveals no established glycosaminoglycan attachment site. One hyaluronan binding motif is present in the predicted sequence of SPACR. We present evidence that SPACR has a functional hyaluronan binding domain, suggesting that interactions between SPACR and hyaluronan may serve to form the basic macromolecular scaffold, which comprises the insoluble interphotoreceptor matrix.

Identification of IHABP, a 95 kDa intracellular hyaluronate binding protein.

The extracellular matrix component hyaluronan is believed to play important roles in various processes of organogenesis, cell migration and cancer. Recognition of and binding to hyaluronan is mediated by cell surface receptors. Three of them, CD44, ICAM-1 and RHAMM (receptor for hyaluronic acid mediated motility), have been identified. A cDNA clone designated RHAMM turned out to possess transforming capacity. Based on this published sequence, we isolated the complete cDNA of the murine gene. The cDNA comprises an open reading frame of 2.3 kb and encodes a 95 kDa protein. The protein carries a hyaluronan binding motif which binds to hyaluronan in vitro but not to heparin or chondroitin sulphate. It is ubiquitously expressed in normal cells and in all tumour cell lines irrespective of their metastatic properties. One tumour cell line, the metastatic Lewis lung carcinoma, expresses a larger 105 kDa variant form of the protein due to a genomic rearrangement. Antibodies raised against the 95 kDa protein were used for subcellular localization studies. The hyaluronan binding protein is not detectable at the cell surface but is rather localized exclusively intracellularly. Clearly, the sequence we have identified encodes a protein with properties substantially different to the RHAMM protein. We tentatively name the protein intracellular hyaluronic acid binding protein, IHABP.

The human hyaluronan receptor RHAMM is expressed as an intracellular protein in breast cancer cells.

The receptor for hyaluronan mediated motility (RHAMM) has been reported to mediate migration, transformation, and metastatic spread of murine fibroblasts. Here we describe the expression of two human RHAMM isoforms, which are generated by alternative splicing of the primary gene transcript, by a series of human breast carcinoma cell lines. A polyclonal antibody, raised against a bacterially expressed RHAMM fusion protein, detected an 85-90 kDa protein by western blot analysis. No correlation between the level of RHAMM mRNA and protein expression with known metastatic/malignant potential of the tumour cell lines was observed. Interestingly, we found that the antibody did not stain the cell surface but the cytoplasm of breast cancer cells. The intracellular localisation of RHAMM was confirmed by subcellular fractionation studies. RHAMM proteins were capable of binding to hyaluronan, but not to heparin or chondroitin sulphate, in an vitro binding assay. We also provide evidence that a potential hyaluronan-binding motif in the N terminus of the protein is not involved in the interaction of RHAMM with hyaluronan. Our findings lead us to conclude that RHAMM does not function as a conventional motility receptor for HA in human breast cancer cells and we suggest the term RHAMM be substituted by 'intracellular hyaluronic acid binding protein' (IHABP).

Molecular Cloning and Characterization of a Putative Mouse Hyaluronan Synthase

We report the isolation of a novel mouse gene which encodes a putative hyaluronan synthase. The cDNA was identified using degenerate reverse transcriptase-polymerase chain reaction. Degenerate primers were designed based upon an alignment of the amino acid sequences of Streptococcus pyogenes HasA, Xenopus laevis DG42, and Rhizobium meliloti NodC. A mouse embryo cDNA library was screened with the resultant polymerase chain reaction product, and multiple cDNA clones spanning 3 kilobase pairs (kb) were isolated. The open reading frame predicted a 63-kDa protein with several transmembrane sequences, multiple consensus phosphorylation sites, and four putative hyaluronan binding motifs. The amino acid sequence displayed 55% identity to mouse HAS, 56% identity to Xenopus DG42, and 21% identity to Streptococcus HasA. Northern analysis identified transcripts of 4.8 kb and 3.2 kb, which were expressed highly in the developing mouse embryo and at lower levels in adult mouse heart, brain, spleen, lung, and skeletal muscle. Transfection experiments demonstrated that mouse Has2 could direct hyaluronan coat biosynthesis in transfected COS cells, as evidenced by a classical particle exclusion assay. These results suggest that mammalian HA synthase activity is regulated by at least two related genes. Accordingly, we propose the name Has2 for this gene.