©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Alternative Exon Splicing within the Amino-terminal Nontriple-helical Domain of the Rat Pro-1(XI) Collagen Chain Generates Multiple Forms of the mRNA Transcript Which Exhibit Tissue-dependent Variation (*)

Julia Thom Oxford , Kurt J. Doege (1) (2), Nicholas P. Morris (1)(§)

From the (1) Research Department, Portland Unit, Shriners Hospital for Crippled Children, Departments of Biochemistry and Molecular Biology and (2) Cell Biology and Anatomy, Oregon Health Sciences University, Portland, Oregon 97201

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Type XI collagen is an integral, although minor component of cartilage collagen fibrils. We have established that alternative exon usage is a mechanism for increasing structural diversity within the amino-terminal non-triple helical domain of the pro-1(XI) collagen gene. cDNA clones spanning the amino-terminal domain were selected from a rat chondrosarcoma library, and were shown to contain two major sequence differences from the previously reported human sequence. The first difference was the replacement of sequence encoding an acidic domain of 39 amino acids in length by a sequence encoding a 51-amino acid basic domain with a predicted pI of 11.9. The second difference was the absence of a sequence that would translate into a highly acidic 85amino acid sequence downstream from the first variation. These two changes, expressed together, result in the replacement of most of the acidic domain with one that is smaller and basic. These two sequence differences serve to identify subdomains of a variable region, designated V1 and V2, respectively. V1a is defined as the acidic 39-amino acid sequence element and V1b is defined as the 51-amino acid basic sequence. Analysis of genomic DNA revealed that both V1a and V1b are encoded by separate adjacent exons in the rat genome and V2 is also encoded in a single exon downstream. Analysis of mRNA from cartilage-derived sources revealed a complex pattern of 1(XI) transcript expression due to differential exon usage. In non-cartilage sources, the pattern is less complex; the most prevalent form is the one containing the two acidic sequences, V1a and V2.


INTRODUCTION

The fibrillar collagens, types I, II, III, V, and XI, combine to form the heterotypic interstitial collagen fibrils common to most tissues (1, 2) . These collagens are related by sequence homology and genomic organization; the defining features of this family of collagens include a major uninterrupted triple-helical domain, flanked by propeptide domains (1, 3, 4, 5) . In cartilage, the bulk of the fibril is composed of type II collagen while type XI contributes a relatively small amount to the mass of the fibril (6) . In a similar way, non-cartilaginous fibrils are mixtures of types I and V or I, III and V, with type I the major component (1) .

The overall sequence and structural similarity among these collagen types can mask specific structural differences which can vary from the subtle to the dramatic. The carboxyl propeptides of collagens are thought to guide the molecular associations leading to formation of a specific subset of possible trimeric molecules from the mixture of fibrillar collagen chains synthesized by the cell; this specificity is likely to be imparted by structural variations among these homologous domains (7) . The type XI heterotrimer is a particularly complex example of this chain selectivity. It is composed of three distinct gene products (1, 2, and 3), where 3(XI) is the same gene product as the 1(II) chain of type II collagen homotrimers (8, 9, 10, 11) . In addition, type V/XI hybrid molecules, with different combinations of the component chains, have been inferred or directly demonstrated in bone (12) , mature articular cartilage (6) , vitreous (13) , and in the molecule formed by the rhabdomyosarcoma cell line A204 (14) . Structural differences have also been observed in the triple-helical domain. Despite a primary structure which does not significantly differ in the overall distribution of key stabilizing amino acids such as proline and hydroxyproline, only collagens V and XI show a complex melting behavior with several stable unfolding intermediates (15, 16) .

The greatest diversity lies in the amino propeptide where dramatic variations in sequence as well as the site and rate of proteolytic processing are observed. The amino propeptides of all of the fibrillar collagen chains contain a minor triple helix separated from the major triple helix by the amino telopeptide. The region of diversity is found between the minor helix and the signal peptide (4, 5) . In the case of 1(I), 1(III), and 2(V), a cystine-rich globular domain comprises most this region (<100 amino acids) and this sequence is lacking in the gene for 2(I). This domain is also found in the gene for 1(II) where it is encoded by exon 2 and is conditionally expressed in a developmentally regulated fashion (17, 18, 19) . A quite different domain is found in the analogous position in the other fibrillar collagen chains, including 1(V), 1(XI), and 2(XI) (20, 21, 22, 23, 24) . Here the globular domain is much larger (>400 amino acids) and more closely related to the amino-terminal noncollagenous domains of the fibril-associated collagens, types IX, XII, and XIV (25) . The sequence contains only four cysteine residues and can be divided into a weakly basic amino-terminal half containing the cysteine residues (designated PARP-like after the pro-2(XI) cleaved domain (23, 24) ) and a very acidic half adjacent to the minor helix (see Fig. 1).


Figure 1: cDNA clones encoding rat pro-1(XI) amino-terminal domain. Overlapping clones were obtained for the amino-terminal domain of 1(XI) from a rat chondrosarcoma cDNA library. Position of clones is compared to general structure of the published amino propeptide; + indicates a slightly basic domain; - - - indicates a strongly acidic region. The numerical scale represents base pairs of DNA, with 0 at the start of translation. Variations in the very acidic region are indicated in Fig. 2. The 5`-most clone (R8E) contains 300 bp of 5`-untranslated sequence. R1-1 and R6D extend into the major triple helix-coding ( TH) region. Signal peptide ( sp), minor helix ( mh), telopeptide ( tp).



In cartilage, the structure of this domain in type XI collagen has been of interest because unlike type II collagen, proteolytic processing of the amino-terminal domain is very slow and even after processing is completed, some portion of the domain is retained (26) . The site(s) of proteolytic cleavage have not been identified. For the pro-1(XI) chain, it is likely that some molecules with an intact amino-terminal domain are incorporated into the growing fibril.

One function of the amino propeptides of fibrillar collagens appears to be an involvement in the process of fibril formation (27) . In vitro, varying the content of pN-collagen() modulates fibrillogensis (28, 29) . In vivo, failure to remove the amino propeptide of type I collagen can result in derangement of the tightly packed cylindrical fibril as seen in dermatosporaxis (28, 30) . The low abundance of type XI collagen, the size of the amino propeptides of type XI collagen, their persistence in the tissue, and the location of the triple helix in the interior of the cartilage fibril suggest that the role of this molecule in fibril formation is more regulatory than structural. Such a role was proposed recently for type V collagen, see also Ref. 21.

The pro-1(XI) amino propeptide was originally cloned and sequenced from a noncartilaginous source (22) . Because the type XI amino propeptide may help regulate formation of collagen fibrils in cartilage, we have cloned and sequenced this domain from a cartilaginous source, rat chondrosarcoma (31) , and looked for structural features specific to this tissue. Our results show that alternative splicing at two sites within the acidic sequence generates multiple forms of the amino-terminal domain. The additional isoforms generated by alternative splicing are quite different from the original noncartilaginous form and, in rat, are more prevalent in cartilage.


MATERIALS AND METHODS

cDNA Cloning and Sequencing

cDNA clones encoding the amino-terminal domain of rat pro-1(XI) were obtained by serial screening of an RCS cDNA library. The library was generated from Swarm rat chondrosarcoma poly(A)RNA (prepared as described previously (32) ), primed for first strand synthesis using both random hexamers and oligo(dT). A modified Gubler and Hoffman (33) method of cDNA construction was used employing kit reagents (Amersham). Double stranded cDNA with EcoRI linkers was ligated to gt11 arms and phage were packaged with commercial extracts (Stratagene). The initial screen utilized a probe obtained by reverse transcriptase-PCR amplification of a fragment of the rat pro-1(XI) carboxyl propeptide (34) from RCS RNA. Primers for amplification were 5`-GGAGAAGTCATACAGCCATTACCT-3` and 5`-CCAAGAAAACAAGCTGGACCAACTTC-3`, and the amplified fragment was cloned and sequenced. DNA fragments were labeled for use as probes with P by random primer extension (Multiprime, Amersham). The 5`-most clone was identified by sequencing of subclones and a new probe from the 5` end prepared by PCR amplification template DNA with primers 5`-TGAGTCCTTGAGGGCCCCC-3` and TAGCCCTGAGTCCTTGAGG-3`. A second round of screening produced clone R1-1 (Fig. 1) identified as the 5`-most by sequencing of the purified phage. A third screen was performed in the same way using a probe generated by PCR with primers 5`-GTGTGGAGAAGAAAACTGTGACAATG and 5`-GTCACAGTCGGGACTGTAATGGTC-3` and R1-1 template DNA. Phage containing the 5`-most cDNA were identified as those yielding the largest products of amplification between the downstream primer used to make the probe and a primer from either side of the gt11 cloning site. Overlapping clones spanning the amino-terminal domain of rat pro-1(XI) (see Fig. 1) were subcloned into pBluescript (Stratagene) and sequenced on both strands. Plasmids were prepared using the alkaline lysis method (35) and purified using DNA Purification Resin (Promega).

DNA Sequencing

Sequencing was performed by the dideoxy chain termination method (36) using either Sequenase DNA polymerase (U. S. Biochemical Corp.) or sequencing grade Taq DNA polymerase and thermal cycle DNA sequencing protocol (fmol, Promega). In some cases, sequences were obtained by automated DNA sequencing using an ABI-373A sequencer (Perkin Elmer). Sequence alignments were performed using programs provided by the Genetics Computer Group (37) .

Cloning of Genomic DNA

A rat genomic DNA library (Clontech) was screened with the ApaI/ EcoRI restriction fragment from clone R1-1 (Fig. 1). Positive plaques were purified and phage DNA grown and prepared as described (35) . EcoRI fragments were screened for exons by Southern hybridization analysis with oligonucleotides from C1, V1b, V2, and C3. Exon-containing fragments were subcloned into pBluescript and sequenced.

Northern Analysis

A probe for V1b was prepared by PCR amplification of cloned template DNA between primers 5`-AAAAGAAATCCAATTACACAAAGA-3` and 5`-GACCCCTAGTTTGGCTTTG-3`. Rat chondrosarcoma poly(A)RNA was probed by Northern analysis as described previously (11) .

RNA Preparation

RNA was extracted from specified tissues of 17-day fetal rat, from a fetal rat skin cell line (FR) (ATCC), and from IRC cells (provided by Walter E. Horton Jr. (38) ), by the method of Chomczynski (39) .

PCR Analysis of Alternatively Spliced Domains

Primer pairs for PCR bracketing variable region V1 were 5`-CCAAGGCAGCATATGACTACTGTG-3` and 5`-GCCGAGGAGACTCAGTCTGG-3` (C1-C2), bracketing variable region V2 were 5`-CCAGACTGAGTCTCCTCGGC-3` and 5`-CATTCCGGGTTCAACTACAGC-3` (C2-C3), and bracketing both V1 and V2 were 5`-CCAAGGCAGCATATGACTACTGTG-3` and 5`-CATTCCGGGTTCAACTACAGC-3` (C1-C3). Primer pairs for glyceraldehyde-3-phosphate dehydrogenase, 5`-GTCAACGGATTTGGCCGTATTGG-3` and 5`-AAAGTTGTCATGGATGACCTTGGCC-3` (40) , were used as a control for PCR. Annealing temperatures were calculated by the PRIMER program (kindly provided by Drs. S. Lincoln, M. Daly, and E. Lander, Massachusetts Institute of Technology, Center for Genome Research). PCR was performed according to established protocols (41) using 1.5 m M MgClin the amplification buffer and 30 cycles of amplification. As a negative control, reverse transcriptase was omitted. PCR products were analyzed by electrophoresis on 3.5% Nusieve 3:1 agarose gels (FMC) and staining with ethidium bromide. Individual amplification products were identified by direct sequencing of gel bands (fmol, Promega) after removal of agarose (PCR clean up resin, Promega). To analyze PCR products without heteroduplex formation, amplification products were end labeled with [-P]ATP (ICN) and polynucleotide kinase (Promega) and electrophoresed on alkaline denaturing gels (35) . Electrophoretic markers, FC 174/ HaeIII digest (Promega), were labeled in the same way.


RESULTS

Screening of the rat chondrosarcoma cDNA library led to the identification of several overlapping clones encoding the amino-terminal domain of pro-1(XI) and some of the 5`-untranslated region (Fig. 1). The sequence from the signal peptide to the start of the major triple-helical domain is presented in Fig. 2. This corresponds to the sequence of clone R6D. In general, the rat amino-terminal domain was very similar to the reported human sequence. The sequence 5` of the central pair of cysteine residues at nucleotides 703 and 724 of the rat sequence showed 87% nucleic acid sequence identity, translating to 93% amino acid similarity. The region of the minor helix and amino telopeptide (nucleotides 1276 to 1615 of the rat sequence) showed 87% identity at the nucleic acid level and 98% similarity at the amino acid level. Between these two regions of high homology lies an area of variability. Two major differences between rat and human were observed in this variable region. Variable region 1 (V1), starting at nucleotide 775 ( shaded area in Fig. 2) was unrelated to the human sequence. Variable region 2 (V2) is the region 93 bases downstream, between 1021 and 1275 ( second shaded area in Fig. 2) which was missing in clones R8E, R6B, and R1-1.


Figure 2: Sequence of cDNA encoding rat 1(XI) amino-terminal domain. Sequence of rat 1(XI) amino-terminal domain is shown in capital letters and numbered with respect to the first base of the start codon. The rat cDNA sequence is compared to that of human; only human DNA sequence different from that of rat is shown in the line of text directly above the rat sequence. The translation of the rat sequence is shown immediately below the rat DNA sequence and differences in human sequence are indicated above the human DNA sequence. Very few differences exist between 1 and 774 and from 927 into the major triple helix, and most result in conservative changes at the amino acid level. An additional codon for serine exists between nucleotides 15 and 16 of the rat sequence and there is an additional codon for proline between nucleotides 738 and 739 of the rat sequence. Shaded area from nucleotide 775 to 927 indicates subdomain 1 of the variable region V, designated V1. The acidic human sequence is termed V1a and the basic rat sequence is termed V1b. Shaded area from 1021 to 1275 indicates subdomain 2 of the variable region, designated V2. The constant region between V1 and V2 is called C2, and the 5`- and 3`-constant regions flanking the variable domains are C1 and C2, respectively. Dashes indicate an absence of nucleotide or amino acid, dots indicate that the nucleotide is the same in both human and rat sequences. Continuous minor helix is underlined. The beginning of the major helix is indicated by solid triangle. The four cysteine residues are boxed.



Inspection of the rat and human deduced amino acid sequence showed that the very acidic (theoretical pI of 3.34) human sequence of 39 amino acids (22) termed V1a was replaced in RCS by a very basic (pI of 11.90) 51-amino acid sequence termed V1b. Northern analysis of RCS poly(A)RNA (data not shown) indicated that this sequence was well represented among RCS pro-1(XI) transcripts. In RCS, the replacement of V1a by V1b, combined with the frequent absence of V2, which also encodes a very acidic sequence, would be predicted to strikingly alter the biochemical nature of this region of the amino-terminal domain. To examine the basis for the differences between the rat and human sequences, a rat genomic library was screened with a cDNA probe specific for this region, the ApaI- EcoRI fragment of cDNA clone R1-1 (Fig. 1). Two non-overlapping genomic clones were obtained and exons were identified and analyzed by sequencing. Sequence data revealed that the V1a domain was also present in the rat gene (Fig. 3). V1a, V1b, C2 (constant region 2), and V2 each occur as separate exons, in this order (Fig. 4). The intron-exon boundaries of the four exons analyzed conform to the consensus splice acceptor and donor sites (42) and do not involve split codons.

The identification of the exons and the differences observed within the rat cDNA sequences and between rat and human forms of 1(XI) pointed to alternative splicing of mRNA as the likely mechanism to generate this diversity. The pattern of exon utilization in various tissues was examined in cDNAs encoding these regions which were amplified by PCR and analyzed by electrophoresis and sequencing. IRC cell, RCS, and 17-day fetal rat sternal cartilage and limb cartilage were used as sources of chondrocyte RNA; placenta and a fetal rat skin cell line were used as sources of noncartilaginous RNA. The V1 region was amplified using primers within the C1 and C2 sequences (Fig. 5 A). Three products which correspond to the presence of V1b (319 bp), the presence of V1a (282 bp), and to the absence of a V1 exon (166 bp) were detected. The identity of these bands was verified by sequencing. All three forms were observed in normal chondrocytes, whereas RCS lacked V1a and IRC lacked V1b. The band just above 319 bp in the IRC lane as well as the uppermost band in RCS are heteroduplexes of the lower two bands. In placenta and skin fibroblasts, only V1a was detected. In only one case was a transcript containing both V1a and V1b observed. Amplification using primers within V1a and C2 (Fig. 5 C) resulted in a larger than expected band only in RCS and this band proved to be V1a + V1b as identified by sequencing. Because V1a is itself rarely expressed in RCS, PCR priming within V1a selects for rare transcripts. Analysis of the region including V2 by amplification between C2 and C3 is shown in Fig. 5B. In all cartilage-derived sources, both splice forms with (364 bp) and without (109 bp) V2 were present. The V2 domain was relatively more abundant in IRC and less so in RCS when compared to normal cartilage. In placenta and skin fibroblasts, only the form with V2 was expressed.


Figure 5: Analysis of exon usage in the V1 and V2 region. PCR amplification reactions were carried out across the variable region of 1(XI) amino-terminal domain. A, three different products are observed for the V1 domain, with V1a, with V1b or minus either form of V1. B, two different products are observed for the V2 domain, using PCR primers within C2 and C3. The identity of these products was determined to be either with or without the V2 exon. C, amplification using primers within V1a and C2 shows a 146-bp product in all tissues, but in addition, a larger product in RCS (297 bp). This band consists of V1a and V1b expressed in the same transcript. Tissues assayed were sterna ( STA), limb ( LMB), rat chondrosarcoma ( RCS), immortalized rat chondrocytes ( IRC), placenta ( PLA), and a fetal rat fibroblast cell line ( FB). Size markers are shown on the left. PCR products were identified by sequencing gel bands. Identity and sizes are shown on the right.



The possibility of coordinate expression of exons from the two domains was examined by PCR amplification across both V1 and V2 using primers in C1 and C3 (Fig. 6 A). A combination of three possibilities with respect to V1 and two for V2 should yield six possible products for this amplification. Six products of the expected sizes were observed as well as additional bands of sizes not predicted from this combination of exons. A simpler picture was obtained when the PCR products were end-labeled and separated on a denaturing alkaline gel (Fig. 6 B), indicating that part of the apparent complexity could be explained by heteroduplex formation between the various products of amplification in cases where more than one product was formed. Only the products expected for combinations of V1 and V2 are present. V0, the splice form lacking both V1 and V2, is well represented in all four chondrocyte-derived RNAs. The form V1a alone is present in IRC but only barely detectable in sterna and limb. V1b was present in normal chondrocytes and RCS but was not observed in IRC. V2 alone and V1a + V2 were found in limb, sterna, and IRC but not in RCS. V1b + V2 was present in low amounts in limb, sterna, and RCS but was absent from IRC. Placenta and skin fibroblasts show only V1a + V2, the form originally cloned and sequenced (22) .


Figure 6: Coordinate expression of exons of V1 and V2. mRNA structure across the entire variable domain was analyzed by amplifying cDNA using PCR primers within C1 and C3. A, ethidium bromide-staining pattern of amplification products. B, autoradiographic image of identical PCR products after end labeling and separating on an alkaline gel. Tissues assayed are as described in the legend to Fig. 5. The two bands below +V1a +V2 in lane FB were not identified. Size markers are shown on the left. Identified products and sizes are shown on the right.



The examination of the pattern of alternative splicing of the variable region was extended to RNA samples from several other noncartilaginous tissues of fetal rat (Fig. 7). Amplification of glyceraldehyde-3-phosphate dehydrogenase was performed on an identical aliquot as a control for the quantity and quality of the RNA to permit a rough estimation of the relative amount of pro-1(XI) transcript in each tissue. Brain, calvaria, skeletal muscle, and skin showed significant expression of pro-1(XI) mRNA.. In each case, the splice form containing V1a + V2 was the prominent species. The splice form lacking both V1 and V2, V0, was also present in calvaria and skeletal muscle as was a low level of +V1b (not visible on this gel). The other tissues, heart, liver, kidney, and lung showed barely detectable levels of pro-1(XI) transcripts and then only the V1a + V2 splice form was visible. Amplification of sternal RNA revealed the same complex pattern of alternative splicing identified above. In all of the PCR amplification experiments presented, no products were amplified when reverse transcriptase was omitted.


DISCUSSION

Alternative exon splicing is now observed to be a relatively common feature of the biosynthesis of extracellular matrix macromolecules including fibronectin, elastin, aggrecan, and some of the collagens (reviewed by Boyd et al. (43) ) and more recently in fibrillin (44) , fibulin 1 (45) , and fibulin 2 (46) , for example. It is clear from such studies that alternative exon splicing can have important functional consequences. Alternative splicing within the exon encoding the V region of fibronectin regulates several properties of the protein (43) . The portion of this exon retained in the fibronectin dimer influences fibronectin secretion from the cell, cell type-specific adhesion, and incorporation of fibronectin into fibrin clots. Within the collagen gene family, alternative splicing can involve triple-helical domains, such as in type XIII, but more generally involves nontriple-helical domains which has been shown for type VI (43) and which has been proposed for types XII and XIV (25, 47) . Recently it was shown that among the fibrillar collagens, the 1(II) gene product can be modulated by alternative splicing of exon 2 which encodes the cysteine-rich domain located between the minor helix and the signal peptide in the amino propeptide (17, 18, 19) . In the absence of direct evidence, functional significance is inferred from the pattern and location of the alternative spliced exons within the mRNA and protein, specific tissue distribution, or specific spatial and temporal pattern of expression during development.

Data presented in this report demonstrate that the rat 1(XI) chain undergoes a complex pattern of alternative exon usage involving two closely associated variable regions, V1 and V2, located between the PARP-like region and the minor helix of the amino-terminal domain. This region, identified from a noncartilaginous source, is highly acidic with a predicted pI of 3.34 (22) . Transcripts from cartilage, analyzed by PCR, indicate that these two variable regions can generate six forms of the amino-terminal domain. These forms arise because in V1 either the V1a exon, or the V1b exon or both can be spliced out while the V2 exon can be either present or absent. These splice isoforms are also observed in RNA from human and chick chondrocyte.Splicing of these exons is coordinated since V1a and V1b are typically expressed in a mutually exclusive fashion; furthermore, V1b expression is usually linked to the absence of V2, V1a to the presence of V2. The V1b exon encodes a very basic amino acid sequence, predicted pI of 11.9, while V1a and V2 are quite acidic. The combinations of these exons observed would produce four prevalent versions of the variable segment of the amino-terminal domain: a basic form, V1b + C2, strongly acidic forms, V1a + C2 + V2 or C2 + V2, and a smaller less acidic form consisting of C2 alone (Fig. 8), designated Vo. The V0 isoform would most closely resemble the structure of the 2(XI) amino-terminal domain (23) . Since C2 is also an acidic sequence and is constitutively expressed, the differences of net charge described among the isoforms are a matter of degree. While it is simplistic to categorize sequences based solely on net charge, the differences are striking enough to be noted.

Embryonic noncartilaginous tissues have been shown to express mRNA transcripts of the 1(XI) gene (48) . Northern analysis of poly(A)RNA with a probe including the carboxyl propeptide coding region showed that chick brain, skin, calvaria, heart, and muscle express transcripts of the 1(XI) collagen gene. Examination of 1(XI) mRNA isoforms in several different rat embryo tissues by PCR showed a similar distribution with the exception of heart. While it is difficult to assess abundance by PCR, use of a standard transcript amplified in parallel provides some basis for comparison. Several other tissues showed negligible levels of 1(XI) transcripts while yielding comparable levels of amplification of transcripts of the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. The correlation of these PCR results with the results from Northern analysis suggests that detection of 1(XI) transcripts in these noncartilaginous tissues is not a trivial result of the extreme sensitivity of PCR. These same tissues also express low levels (relative to cartilage) of 1(II) (49) and 2(XI) mRNA (50) . Expression of 1(XI) in these noncartilaginous tissues also shows a degree of specificity. The acidic form, V1a + C2 + V2, is prominent in the type XI-positive tissues, consistent with the splice form of the amino-terminal domain initially cloned and sequenced in human placenta and other sources (22) . Rat placenta also expresses this form primarily. Skin and calvaria may also express small amounts of V1b + C2 and V0. Northern analysis using total RNA, conditions selective for more abundant transcripts, indicates that the 1(XI) gene is transcribed more extensively only in cartilage and tendon (58) . Surprisingly, splice forms with V1b but not V1a were detected in tendon.

Within cartilage, all six isoforms of the amino-terminal domain are represented. Relative abundance cannot be determined accurately by PCR. However, end labeling and analysis on denaturing alkaline gels represents each isoform on a molar rather than mass basis so that the observation of large differences in signal may be relevant. Significant among the isoforms are the basic version, V1b + C2, and Vo. It is not known if a single chondrocyte expressing 1(XI) synthesizes all of these forms or whether splice patterns correlate with degree of differentiation or some other aspect of chondrocyte biology. In this regard, it is interesting to note that the two transformed chondrocytes examined, RCS and IRC, each express a subset of the isoforms. IRC cells synthesize only the Vo and the acidic forms, while RCS cells synthesize predominantly Vo and the basic form. This could reflect some developmental or inherent property of chondrocytes or it may just reflect an aberration resulting from transformation. At the protein level, we have previously observed multiple electrophoretic forms of pro-1(XI) synthesized by metabolically labeled chick sternal chondrocytes (26) and IRC cells (11) . It is likely that these may represent different splicing isoforms, although this has yet to be demonstrated.

The 51-amino acid peptide encoded by the V1b exon is a unique sequence based on comparison to the sequence data base. The sequence is unusual in that a majority of the basic residues (mostly lysines) are found in clusters of three, four, or five residues. Such clusters might serve as sites for proteolysis and generate a more readily processed form of the 1(XI) amino-terminal domain. In some cases the clusters are separated by four or six amino acids in which case they fit the putative hyaluronan binding motif, B(X7)B, of Yang et al. (51) , where B represents a basic amino acid and X denotes any non-acidic amino acid.

In addition to the alternative splicing reported here for the 1(XI) chain, essentially the same regions of the 2 (XI)and 3(XI) ( i.e. 1(II)) undergo alternative splicing events. The sequence of the variable region of 1(XI) is unrelated to the analogous region of 1(V) while the remainder of the two chains is very homologous. The sequence of this region of 1(V) also shows significant species or tissue-dependent variation (20, 21) , although no evidence of alternative exon usage in this region has been reported.

Fetal and growth plate cartilage collagen fibrils are relatively small in diameter, less than 25 nm. As a minor but integral component of these fibrils (2, 52) , type XI collagen has been proposed as an element involved in the regulation of fibril diameter. Evidence for this function includes: very slow proteolytic processing within the pro-1(XI) amino-terminal domain (26) whose large size would not likely be accommodated within the fibril; limitation of cartilage fibril diameter when fibrils were reconstituted with the matrix form of type XI (53) ; demonstration by immunoelectron microscopy of the PARP-like domain at the surface of thin but not thick fibrils in cartilage growth plates (54) ; and finally, failure of the homozygous recessive chondrodystrophic mouse ( CHO) to synthesize the 1(XI) chain (55) , resulting in cartilage which contains unusually large fibrils and fails to retain proteogylcans effectively (56, 57) .

Based on structural data, the proteolytic processing site(s) likely resides between the PARP-like domain and the minor helix (23, 24, 27) . This is precisely the location of the variable region, portions of which could be retained after processing. The PARP-like domains and the variable regions could mediate interactions directly or indirectly between the proteoglycan network and the collagen fibrils. The alternatively spliced exons, V1b, V1a, and V2 could influence fibril formation and interactions by modulating processing of the PARP-like domain or, independent of processing, could, in themselves, provide sites for interaction with the cartilage matrix.


FOOTNOTES

*
This work was supported by grants from the Shriners Hospital for Crippled Children and by Grant GM-39862 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U20116-U20121.

§
To whom correspondence should be addressed: Research Department, Portland Unit, Shriners Hospital for Crippled Children, 3101 S.W. Sam Jackson Pk. Rd., Portland, OR 97201. Tel.: 503-221-1537; Fax: 503-221-3451; E-mail: NPM@SHCC.org.

The abbreviations used are: pN-collagen, an intermediate in the proteolytic processing of procollagen retaining an amino propeptide but lacking the carboxyl propeptide; PCR, polymerase chain reaction; RCS, Swarm rat chondrosarcoma; IRC, immortalized fetal rat chondrocytes; PARP, proline/arginine-rich protein; bp, base pair(s).


ACKNOWLEDGEMENTS

We thank Silvija Coulter, Babette Romancier, and Xiaocun Chen for expert technical assistance. We also acknowledge Jay Gambee and Jeff Bondar of the analytical core facility, Shriners Hospital, Portland, OR, for the provision of automated sequencing and oligonucleotides and Rich Watson for assistance with computer programs and computer graphics.


REFERENCES
  1. Burgeson, R. E. (1988) Annu. Rev. Cell Biol. 4, 551-577 [CrossRef]
  2. Mendler, M., Eich-Bender, S. G., Vaughan, L., Winterhalter, K. H., and Bruckner, P. (1989) J. Cell Biol. 108, 191-197 [Abstract]
  3. Kuhn, K. (1987) in Structure and Function of Collagen Types (Burgeson, R. E., and Mayne, R., eds) pp. 1-42, Academic Press, Orlando, FL
  4. Ramirez, F., Boast, S., D'Alessio, M., Lee, B., Prince, J., Su, M.-W., Vissing, H., and Yoshioka, H. (1990) Ann. N. Y. Acad. Sci. 580, 74-80 [Medline] [Order article via Infotrieve]
  5. Sandell, L. J., and Boyd, C. D. (1990) in Extracellular Matrix Genes (Sandell, L. J., and Boy, C. D., eds) pp. 1-56, Academic Press, New York
  6. Eyre, D., and Wu, J.-J. (1987) in Structure and Function of Collagen Types (Mayne, R., and Burgeson, R. E., eds) pp. 81-103, Academic Press, Orlando, FL
  7. Lees, J. F., and Bulleid, N. J. (1994) J. Biol. Chem. 269, 24354-24360 [Abstract/Free Full Text]
  8. Morris, N. P., and Bachinger, H. P. (1987) J. Biol. Chem. 262, 11345-11350 [Abstract/Free Full Text]
  9. Burgeson, R. E., and Hollister, D. W. (1979) Biochem. Biophys. Res Commun. 87, 1124-1131 [Medline] [Order article via Infotrieve]
  10. Furuto, D. K., and Miller, E. J. (1983) Arch. Biochem. Biophys. 226, 604-611 [Medline] [Order article via Infotrieve]
  11. Oxford, J. T., Doege, K. J., Horton, W. E., Jr., and Morris, N. P. (1994) Exp. Cell Res. 213, 28-36 [CrossRef][Medline] [Order article via Infotrieve]
  12. Niyibizi, C., and Eyre, D. R. (1989) FEBS Lett. 242, 314-318 [CrossRef][Medline] [Order article via Infotrieve]
  13. Mayne, R., Brewton, R. G., Mayne, P. M., and Baker, J. R. (1993) J. Biol. Chem. 268, 9381-9386 [Abstract/Free Full Text]
  14. Kleman, J.-P., Hartmann, D. J., Ramirez, F., and van der Rest, M. (1992) Eur. J. Biochem. 210, 329-335 [Abstract]
  15. Morris, N. P., Watt, S. L., Davis, J. M., and Bachinger, H. P. (1990) J. Biol. Chem. 265, 10081-10087 [Abstract/Free Full Text]
  16. Bächinger, H. P., and Davis, J. M. (1991) Int. J. Biol. Macromol. 13, 152-156 [CrossRef][Medline] [Order article via Infotrieve]
  17. Ryan, M. C., and Sandell, L. J. (1990) J. Biol. Chem. 265, 10334-10339 [Abstract/Free Full Text]
  18. Sandell, L. J., Morris, N. P., Robbins, J. R., and Goldring, M. B. (1991) J. Cell Biol. 114, 1307-1319 [Abstract]
  19. Sandell, L. J., Nalin, A. M., and Reife, R. A. (1994) Dev. Dynamics 199, 129-140 [Medline] [Order article via Infotrieve]
  20. Greenspan, D. S., Cheng, W., and Hoffman, G. G. (1991) J. Biol. Chem. 266, 24727-24733 [Abstract/Free Full Text]
  21. Linsenmayer, T. F., Gibney, E., Igoe, F., Gordon, M. K., Fitch, J. M., Fessler, L. I., and Birk, D. E. (1993) J. Cell Biol. 121, 1181-1189 [Abstract]
  22. Yoshioka, H., and Ramirez, F. (1990) J. Biol. Chem. 265, 6423-6426 [Abstract/Free Full Text]
  23. Zhidkova, N. I., Brewton, R. G., and Mayne, R. (1993) FEBS Lett. 326, 25-28 [CrossRef][Medline] [Order article via Infotrieve]
  24. Neame, P. J., Young, C. N., and Treep, J. T. (1990) J. Biol. Chem. 265, 20401-20408 [Abstract/Free Full Text]
  25. Walchli, C., Trueb, J., Kessler, B., Winterhalter, K. H., and Trueb, B. (1993) Eur. J. Biochem. 212, 483-490 [Abstract]
  26. Thom, J. R., and Morris, N. P. (1991) J. Biol. Chem. 266, 7262-7269 [Abstract/Free Full Text]
  27. Chapman, J. A. (1989) Biopolymers 28, 1367-1382 [Medline] [Order article via Infotrieve]
  28. Holmes, D. F., Watson, R. B., Steinmann, B., and Kadler, K. (1993) J. Biol. Chem. 268, 15758-15765 [Abstract/Free Full Text]
  29. Romanic, A. M., Adachi, E., Kadler, K. E., Hojima, Y., and Prockop, D. J., (1991) J. Biol. Chem. 266, 12703-12709 [Abstract/Free Full Text]
  30. Pièrard, G. E., La, T., Hermanns, J.-F., Nusgans, B. V., and Lapiere, C. M. (1986) Collagen Rel. Res. 6, 481-492
  31. Smith, B. D., Martin, G. R., Miller, E. J., Dorfman, A., and Swarm, R. (1975) Arch. Biochem. Biophys. 166, 181-186 [Medline] [Order article via Infotrieve]
  32. Doege, K., Sasaki, M., Horigan, E., Hassell, J. R., and Yamada, Y. (1987) J. Biol. Chem. 262, 17757-17767 [Abstract/Free Full Text]
  33. Gubler, U., and Hoffman, B. J. (1983) Gene ( Amst) 25, 263-269 [Medline] [Order article via Infotrieve]
  34. Bernard, M., Yoshioka, H., Rodriguez, E., van der Rest, M., Kimura, T., Ninomiya, Y., Olsen, B. R., and Ramirez, F. (1988) J. Biol. Chem. 263, 17159-17166 [Abstract/Free Full Text]
  35. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  36. Sanger, F., Nicklin, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  37. Genetics Computer Group (1991) Program Manual for the GCG Package, Version 7, April 1991, Genetics Computer Group Inc., Madison, WI
  38. Horton, W. E., Jr., Rapp, C. J., Nemuth, U., Bolander, M., Doege, K. J., Yamada, Y., and Hassell, J. R. (1988) Exp. Cell Res. 178, 457-468 [Medline] [Order article via Infotrieve]
  39. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  40. Tso, J. Y., Sun, X.-H., Kao, T.-H., Reece, K. S., and Wu, R. (1985) Nucleic Acids Res. 13, 2485-2502 [Abstract]
  41. Ausubel, F. M., Brent, R, Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1990) Current Protocols in Molecular Biology, Wiley and Sons New York
  42. Sharp, P. A. (1981) Cell 23, 643-646 [Medline] [Order article via Infotrieve]
  43. Boyd, C. D., Pierce, R. A., Scharzbauer, J. E., Doege, K. J., and Sandell, L. J. (1993) Matrix 13, 457-469 [Medline] [Order article via Infotrieve]
  44. Corson, G. M., Chalberg, S. C., Dietz, H. C., Charbonneau, N. L., and Sakai, L. Y. (1993) Genomics 17, 476-484 [CrossRef][Medline] [Order article via Infotrieve]
  45. Pan, T.-C., Kluge, M., Zhang, R.-Z., Mayer, U., Timpl, R., and Chu, M.-L. (1993) Eur. J. Biochem. 215, 733-740 [Abstract]
  46. Zhang, R.-Z., Pan, T.-C. Zhang, Z.-Y., Mattei, M.-G., Timpl, R., and Chu, M.-L. (1994) Genomics 22, 425-430 [CrossRef][Medline] [Order article via Infotrieve]
  47. Trueb, J., and Trueb, B. (1992) Biochim. Biophys. Acta 1171, 97-98 [Medline] [Order article via Infotrieve]
  48. Nah, H.-D., Barembaum, M., and Upholt, W. B. (1992) J. Biol. Chem. 267, 22581-22586 [Abstract/Free Full Text]
  49. Nah, H.-D., and Upholt, W. B. (1991) J. Biol. Chem. 266, 23446-23452 [Abstract/Free Full Text]
  50. Sandberg, M. M., Hirvonen, H. E., Elima, K. J. M., and Vuorio, E. I. (1993) Biochem. J. 294, 595-602 [Medline] [Order article via Infotrieve]
  51. Yang, B., Yang, B. L., Savani, R. C., and Turley, E. A. (1994) EMBO J. 13, 286-296 [Abstract]
  52. Petit, B., Ronzier, M. C., Hartmann, D. J., and Herbage, D. (1993) Histochemistry 100, 231-239 [Medline] [Order article via Infotrieve]
  53. Eikenberry, E. F., Mendler, M., Bürgin, R., Winterhalter, K. H., and Bruckner, P. (1992) in Articular Cartilage and Osteoarthritis (Kuettner, K. E., Schleyerbach, R., Peyron, J. G., and Hascall, V. C., eds) pp. 133-149, Raven Press, New York
  54. Keene, D. R., Oxford, J. T., and Morris, N. P. (1993) Mol. Biol. Cell 4, 289a
  55. Li, Y., Lacerda, D. A., Warman, M., Beier, D., Oxford, J. T., Morris, N. P., Andrikopoulos, K., Ramirez, F., Taylor, B., Seegmiller, R., and Olsen, B, R. (1995) Cell 80, 423-430 [Medline] [Order article via Infotrieve]
  56. Seegmiller, R., Fraser, F. C., and Sheldon, H. (1971) J. Cell Biol. 48, 580-593 [Abstract/Free Full Text]
  57. Seegmiller, R., Ferguson, C. C., and Sheldon, H. (1972) J. Ultrastruct. Res. 38, 288-301 [Medline] [Order article via Infotrieve]
  58. Zhidkova, N. I., Justice, S. K., and Mayne, R. (1995) J. Biol. Chem. 270, 9486-9493 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.