Molecular Vision 2000; 6:148-156 <http://www.molvis.org/molvis/v6/a20/> Received 26 June 2000 | Accepted 17 August 2000 | Published 24 August 2000

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Molecular characterization of the murine orthologue of the human retinal proteoglycan IPM 150

Markus H. Kuehn, David T. Wietecki, Gregory S. Hageman
 
 

Center for Macular Degeneration, Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA

Correspondence to: Gregory S. Hageman, Ph.D., Center for Macular Degeneration, Department of Ophthalmology and Visual Sciences, The University of Iowa, 11190E PFP, 200 Hawkins Drive, Iowa City, IA, 52240; Phone: (319) 384-9722; FAX: (319) 335-7142; email: gregory-hageman@uiowa.edu

Abstract

Purpose: We recently identified a family of novel human proteoglycans/glycoproteins that are major constituents of the human interphotoreceptor matrix. Two members of this family, designated IPM 150 and IPM 200, have been extensively characterized. Although the IPM is thought to mediate crucial roles in retinal physiology, including retinal adhesion and photoreceptor cell viability, little is known about the roles of specific IPM constituents in these processes. In order to characterize the mouse IPM 150 orthologue, to initiate functional in vivo studies, and as a prerequisite towards future genetic manipulation, we cloned the murine orthologue of human IPM 150 and determined its chromosomal location.

Methods: A mouse retinal cDNA library was screened using an IMAGE clone with sequence similarity to human IPM 150. The genomic location of the mouse IPM 150 gene was determined by radiation hybrid analyses.

Results: We describe here the molecular structure of the murine orthologue of human IPM 150 and place the location of its gene on mouse chromosome 9. Among the tissues examined, expression of IPM 150 appeared to be restricted to the retina.

Conclusions: Comparison of the human and murine IPM 150 core proteins revealed that the molecules are generally well conserved, although several potentially significant differences do exist. In addition, two highly conserved domains within the core proteins were identified. The data presented here represent a first step towards the development of experimental murine models, which may eventually be used to elucidate the mechanisms underlying retinal adhesion and photoreceptor survival.

Introduction

Extracellular matrix constituents play crucial roles in neuronal survival and determination, cell migration and synapse formation (reviewed in [1-5]). The outer segments and ellipsoids of mammalian retinal photoreceptor cells are encapsulated by an extracellular matrix referred to as the interphotoreceptor matrix (IPM) [6-10]. The IPM is unique compositionally in that it lacks most molecules that are typically associated with extracellular matrices in other tissues such as collagens (reviewed in [9]). Instead, it is composed of large mucin-like glycoproteins, hyaluronic acid, proteoglycans, and a host of smaller proteins, such as interphotoreceptor retinoid binding protein (IRBP) and various growth factors [11-14].

The distribution of some IPM-associated molecules is not homogenous; discrete subdomains that are rich in distinct IPM constituents have been identified [15-17]. For example, cone photoreceptor cell-associated, but not rod photoreceptor cell-associated, IPM is bound by antibodies directed against chondroitin 6-sulfate and peanut agglutinin (PNA). These domains have been termed "cone matrix sheaths" (CMS) [18,19]. The existence of CMS has been reported in all species of mammals examined thus far [15-17,19-26]. However, variations in the distribution of anti-chondroitin 6-sulfate antibody-binding IPM constituents exist. For example, while these glycosaminoglycans appear to be restricted to CMS in primates, immunohistochemical analyses of rodent retinae have suggested that chondroitin 6-sulfate is present throughout the IPM where it is concentrated immediately adjacent to the apical surfaces of the retinal pigment epithelium (RPE) and neural retina that border the subretinal space [16,27].

The cDNAs encoding two chondroitin sulfate proteoglycans of human IPM, designated IPM 150 (AF047492) and IPM 200 (AF173155), have been cloned previously in this laboratory [28-30]. The cDNA sequences encoding these two molecules have been verified by other investigators and renamed SPACR and SPACRCAN [31,32]. Together, IPM 150 and IPM 200, and their related isoforms, comprise a novel family of extracellular proteins, that are highly expressed by retinal photoreceptor cells [29,30].

A number of studies have demonstrated the involvement of CMS constituents in retinal adhesion, metabolic transport, growth factor sequestration and photoreceptor survival [9,11,25,27,33,34]. Other suspected roles for IPM constituents include transport of metabolites across the subretinal space and mediation of photoreceptor cell outer segment alignment. However, no direct evidence for the involvement IPM 150 or IPM 200 in these processes yet exists. Investigations into the physiological function of these molecules have been hampered largely by the lack of a manipulatable experimental animal model. In order to determine whether an IPM 150 orthologue is expressed in the mouse retina, to provide the probes necessary to initiate in vivo studies into the function of IPM 150, and as a first step towards future genetic manipulation, we have cloned and sequenced the murine orthologue of human IPM 150 and determined its chromosomal location.

Methods

cDNA library screening

IMAGE clone 577006 was used to screen a mouse retinal cDNA library in lambda ZAP (a gift from Dr. Muna Naash, University of Oklahoma) by plaque hybridization. Screening of 200,000 plaques identified three cDNA clones which appeared to contain the desired sequences. The cDNA inserts of these clones (designated 151.1.1; 152.1.2 and 164.3.1) were subcloned by in vivo excision and their sequences were determined by fluorescent cycle sequencing on an ABI model 377 sequencer (Perkin Elmer, Foster City, CA).

5' RACE

Following elucidation of the cDNA sequences of clones 151.1.1, 152.1.2 and 164.3.1, several anti-sense PCR primers were designed for the purpose of amplifying the remaining 5' regions of the corresponding cDNAs. Using RNA-ligase mediated 5' RACE (RLM-RACE, Ambion Inc., Austin, TX), a PCR adapter was ligated to the decapped 5' ends of mouse retinal polyA⁺ molecules. Following reverse transcription, the cDNA was amplified with a pair of nested anti-sense primers (AS#1: 5'- CTC CGA ATC CTG GAA GTT TC -3'; AS#2: 5'- TGT GGT TTG GCA GAG AGA TG -3') that were designed based upon the cDNA sequence of clone 164.3.1 and two sense primers homologous to the adapter molecule. The resulting amplicons were cloned into the pCR 2.1 vector (Invitrogen, San Diego, CA) and their sequences were determined.

Northern blot analyses

Total RNA was isolated from mouse retina, kidney, lung and thymus using the RNeasy system (Qiagen, Valencia, CA). Prior to electrophoresis the samples were denatured at 55 °C for 15 min in 6.5% formaldehyde and 50% formamide in MOPS running buffer (40 mM morpholinopropanesulfonic acid, pH 7.0, containing 100 mM sodium acetate and 10 mM EDTA). The RNA was then fractionated on agarose gels containing formaldehyde and subsequently transferred to nylon-based membranes by capillary transfer using 20X SSC (3 M NaCl and 0.3 M sodium citrate). RNA was cross-linked to the membranes and hybridized with ³²P-labeled DNA probes (derived from clone 164.3.1) for detection of transcripts.

Radiation hybrid analysis

The T31 radiation hybrid panel (Research Genetics, Huntsville, AL) was employed for radiation hybrid mapping. Sense (5'- CAT GCT TGA ACA AGA GC -3') and anti-sense (5'- AAG CTA ACC TTA TCT TTG C -3') primers were designed according to nucleotide sequences 2,746 through 2,763 and 2,879 through 2,896, respectively, of mouse IPM 150. Samples were denatured for 5 min, at 94 °C, and amplified for 35 cycles under the following conditions: 94 °C for 30 s, 50 °C for 30 s, 72 °C for 30 s, in a DNA thermocycler. After amplification, 5 ml of stop solution (95% formamide, 10 mM NaOH, 0.05% Bromophenol Blue, 0.05% Xylene Cyanol) were added to each sample. Amplification products were evaluated by electrophoresis and the data submitted to the Jackson Laboratory Webserver for computation of the chromosomal localization of the gene.

Results

Characterization of mouse IPM 150

Comparison of sequences contained in the GenBank databases to that of the human IPM 150 cDNA sequence revealed the existence of several IMAGE consortium mouse expressed sequence tag (ESTs) clones that displayed homology to the 3' region of human IPM 150. Screening of a mouse retinal cDNA library, using clone 577006 as a probe, resulted in the identification of three cDNA clones that were isolated and sequenced. These clones contained cDNA inserts of 2.7 kb, 1.85 kb and 1.86 kb and the sequences of their overlapping regions were identical. However, none of them encoded sequences analogous to the NH₂-terminus of human IPM 150 [29], suggesting that the full length cDNA sequence had not been obtained. The remaining 5' sequences were obtained with the use of RLM-RACE [35]. Using this approach we generated a PCR fragment of 789 bp, designated MM-1, that encoded the 5' end of mouse IPM 150. Sequence analyses revealed that the 3' region of MM-1 is homologous to sequences obtained from the 5' region of clone 164.3.1 (Figure 1). Computational assembly of all obtained cDNA sequences resulted in the complete cDNA sequence for mouse IPM 150 presented here (Figure 2). Translation of the open reading frame demonstrated that the encoded protein was comprised of 798 amino acids and that it had a predicted molecular weight of 89.5 kDa and an isoelectric point of 4.9.

Comparison of mouse IPM 150 and human IPM 150

Alignment of the primary amino acid sequences of mouse IPM 150 and human IPM 150 indicated that IPM 150 was well conserved between these two species (Figure 3). Amino acid sequence conservation was particularly pronounced in the NH₂- and COOH-terminal regions of the protein. As is the case for its human orthologue, both the NH₂-terminal and the COOH-terminal domains of mouse IPM 150 contained numerous cysteine residues that likely constituted an EGF-like domain in the carboxy terminal region [36]. Furthermore, both molecules possessed an NH₂-terminal hydrophobic domain, which in all likelihood represented a signal peptide. No additional hydrophobic regions were present (Figure 4), suggesting that mouse IPM 150, like its human orthologue, is secreted into the extracellular space and does not remain membrane associated. Mouse IPM 150 contained five consensus sequences for N-linked and over twenty putative sites for attachment of O-linked oligosaccharides [37]. In addition, the mouse IPM 150 core protein possessed three serine-glycine dipeptides and two D/EGS motifs for potential glycosaminoglycan (GAG) attachment [38,39]. Consensus sequences for N- and O-linked carbohydrate attachment were separated spatially along the core protein. Sites of potential N-linked carbohydrate attachment were restricted to the amino and carboxy terminal domains, whereas all sites suitable for O-linked glycosylation and GAG attachment were clustered in a proline rich mucin-like central domain, located between Ala¹⁷⁷ and Gly⁵⁷⁴ (Figure 2).

Previous comparison of human IPM 150 and IPM 200 [30] suggest that two regions of their core proteins are conserved among the members of this protein family. Analyses revealed that these domains, designated C1 and C2, were also highly conserved in mouse IPM 150 (Figure 5 and Figure 6).

Expression of IPM 150 in the mouse

Northern blot analysis of total RNA derived from mouse retina, lung, thymus and kidney revealed the presence of two transcripts of 4.7 and 5.3 kb in the retina that hybridized with the mouse IPM 150 probe (Figure 7).

Genomic localization

Radiation hybrid analyses indicated that the mouse IPM 150 gene was located on murine chromosome 9, between markers D9Mit264 and D9Mit265. This locus lies 42-43 centimorgans from the centromere of mouse chromosome 9 and is syntenic to human chromosome 6q12-13, in good agreement with the genomic localization determined for the human IPM 150 gene, IMPG1 [40,41].

Discussion

Numerous reports have focused upon the structure, composition and function of the IPM (reviewed in [9]). Taken together, these studies provide convincing data that supports the functional importance of IPM constituents [16,21,33,34,42,43]. However, insight into the precise functional role(s) of specific IPM components, including IPM 150 and IPM 200, is generally lacking.

The recent molecular characterization of the core proteins of two of the major proteoglycans of the human IPM, designated IPM 150 and IPM 200 [28-30], represents a significant advance towards our goal of identifying the functions of IPM constituents. Manipulative experimentation aimed at functional characterization of these photoreceptor specific proteins would likely proceed at a more rapid pace if conducted in mice or rats. In order to facilitate this approach we have characterized the mouse IPM 150 cDNA sequence and determined the chromosomal location of its gene.

Comparison of the mouse IPM 150 amino acid sequence to its human orthologue revealed that the overall structure of this protein was conserved across phylogenetic lines. Sequence conservation was particularly pronounced in the amino and carboxy terminal regions of the two core proteins. Consensus sequences for N-glycosylation were clustered in these terminal domains, similar to that observed in human IPM 150 and IPM 200. Interestingly, numerous sites for potential carbohydrate attachment exist in the central regions of both human and mouse IPM 150 [37], even though amino acid sequence conservation is relatively low in this domain. Furthermore, mouse IPM 150 contained an EGF-like domain, as well as several potential hyaluronic acid binding domains [44], features also observed in human IPM 150.

Potentially significant differences between human and mouse IPM 150 do exist. Most strikingly, mouse IPM 150 contained three serine-glycine dipeptides, as well as two D/E GS sequences, motifs that serve as attachment sites for glycosaminoglycan (GAG) moieties [38,39,45]. In contrast, only one potential GAG attachment site is present in the human IPM 150 core protein. These data indicated that mouse IPM 150 might possess more GAG chains than human IPM 150, although neither the extent to which any of the attachment sites are utilized, nor details pertaining to the length(s) and/or composition of IPM 150-associated glycans have been elucidated.

Northern blot analyses indicated that, among the tissues examined, expression of mouse IPM 150 was restricted to the retina. Two differently sized IPM 150 transcripts have been identified. These transcripts may correspond to isoforms of mouse IPM 150 or, alternatively, one of the observed signals could be due to crosshybridization of the probe to a related transcript such as IPM 200 which, in human, displays regional homology to IPM 150 [30]. The absence of transcripts in the thymus-derived RNA sample was unexpected, since several expressed sequence tags (ESTs) exist that share homology to IPM 150 and were derived from mouse thymoidal tissue. Since Northern blot analysis may not detect sequences expressed at low levels, we employed more sensitive RT-PCR assays to assess IPM 150 expression in the mouse thymus. Again, IPM 150 transcripts could only be amplified from retinal cDNA (data not shown), introducing the possibility that the EST sequences contained within GenBank are derived from contaminating genomic DNA.

Based on previous studies supporting a role for IPM proteoglycans in retinal adhesion [21,25,34,46], we propose that IPM 150 and IPM 200 may effect retinal adhesion between the photoreceptor outer segments and the RPE. In this model, the membrane bound isoforms of IPM 150 or IPM 200 would serve as retinal anchors. Secreted forms of IPM 150 or IPM 200 might function to link the retinal anchor(s) to receptors on the surface of the RPE. The vitronectin receptor has been localized to the surfaces of photoreceptor outer segments and the apical microvilli of RPE cells [47] and might serve as a ligand for the IPM proteoglycans. Although IPM 150 and IPM 200 do not possess any RGD consensus sequences, a potential interaction between their core proteins and integrin receptors is not precluded [48,49]. In addition, the chondroitin sulfate GAGs of these IPM proteoglycans might participate in retinal adhesion through receptors like CD44 [50], an adhesion molecule associated with Müller cell apical microvilli in mice and humans (see [9]). Involvement of chondroitin sulfate proteoglycans in retinal adhesion is consistent with data derived from in vivo studies documenting disruption of retinal adhesion following subretinal administration of glycolytic enzymes or perturbation of chondroitin sulfate glycosaminoglycan biosynthesis [25,33,34,51]. Alternatively, the amino terminal cysteine residues may function to link several core proteins together to form an extensive network of matrix molecules. This may explain the relative insolubility of the IPM in aqueous solutions [8,19,52-56].

The presence of numerous hyaluronan (HA) attachment motifs within the IPM 150 core protein has been interpreted as an indication that IPM 150 and HA may form extensive networks to provide the primary scaffold of the IPM [57]. Early biochemical characterizations of the IPM suggested that some of the glycosaminoglycans in the subretinal space may be hyaluronate (HA) [58,59]. More recent histochemical studies, using biotinylated tryptic peptides of link protein and aggrecan as probes, have presented data suggesting that the IPM of adult human and various mammalian eyes may contain HA [22,60,61]. In these studies HA appears to be distributed throughout the IPM, in all species examined except in mice, but may be absent from the space occupied by CMS. Unfortunately, the material labeled by the probe utilized is highly resistant to Streptomyces hyaluronidase digestion. This enzyme, in contrast to testicular hyaluronidase, which also degrades chondroitin sulfate, is highly specific for HA [62,63]. To demonstrate specific binding of the probe, the authors pre-incubated the molecule with purified HA, which abolished binding to IPM material. However, this "control" merely demonstrates that the probe does bind HA, and makes no statements regarding its substrate specificity. The presence of HA in the subretinal space is further put into question by experiments demonstrating that subretinal injections of Streptomyces hyaluronidase, in contrast to testicular hyaluronidase, do not alter the structure or PNA binding pattern of CMS or induce retinal detachments in rabbits [51]. Thus the presence of HA in the IPM and its potential function remain uncertain.

The data presented here, when combined with previous studies [29,30], support the contention that IPM 150 and IPM 200, and their related isoforms, constitute a new family of glycoconjugates. For example, comparison between the amino acid sequences of mouse and human IPM 150, human IPM 200, and the rat IPM 200 orthologue PG 10.2 [64], reveals the presence of two conserved domains within their core proteins. These domains, designated C1 and C2, are located within the amino- and carboxy- termini of the core proteins, respectively, and display a significant degree of sequence conservation. These regions do not display any sequence homology to previously described proteins and their function(s) is, as of yet, unclear. However, the high degree of sequence conservation indicates potential functional significance of these domains.

Complete knowledge of the IPM molecules that elicit retinal adhesion and photoreceptor survival will be of paramount importance for understanding the biology of the IPM in pathologic processes such as retinal detachment and photoreceptor degeneration. The data presented herein represent a first step towards the development of experimental murine models which may eventually lead to the establishment of intervention modalities in the future.

Acknowledgements

The authors would like to thank Dr. Bo Chang for assistance with the radiation hybrid analysis, Dr. Muna Naash for the gift of the mouse retinal cDNA library, Dr. Val Sheffield and his staff for assistance during the sequencing of the obtained cDNA clones, and Dr. Robert Mullins for helpful discussion throughout the project. Supported in part by NIH EY06463, a Research to Prevent Blindness Lew R. Wasserman Merit Award, and an unrestricted grant to the Department of Ophthalmology and Visual Sciences from Research to Prevent Blindness, Inc.

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